1-tetrahydropyranylcarbonyl-2,3-dihydro-1H-indole compounds for treating cancer

ABSTRACT

The present invention relates to certain novel 2,3-dihydro-1H-indole compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds to treat cancer, more particularly for the treatment of cancer selected from the group consisting of melanoma, acute myeloid leukemia, chronic lymphocytic leukemia, colorectal cancer, breast cancer, lung cancer, ovarian cancer, fallopian tube carcinoma, primary peritoneal carcinoma, cervical cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, glioblastoma, non-Hodgkin&#39;s lymphoma, and Hodgkin&#39;s lymphoma.

The present invention relates to novel 2,3-dihydro-1H-indole compoundsthat inhibit the conversion of tryptophan to kynurenine, certain ofwhich have been confirmed to bind to indoleamine 2,3-dioxygenase (IDO1).The present invention also relates to pharmaceutical compositionscomprising these compounds and methods of using these compounds to treatphysiological disorders, more particularly for the treatment of cancersuch as melanoma, acute myeloid leukemia, chronic lymphocytic leukemia,colorectal cancer, renal cell carcinoma, breast cancer, lung cancer,ovarian cancer, fallopian tube carcinoma, primary peritoneal carcinoma,cervical cancer, gastric cancer, liver cancer, pancreatic cancer,thyroid cancer, glioma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

Tryptophan is an essential amino acid required for protein biosynthesis,cellular growth, the generation of neuroactive metabolites such asserotonin (5-hydroxytryptamine), melatonin, and the co-enzymenicotinamide adenine dinucleotide (NAD). Tryptophan is catabolized byindoleamine 2,3-dioxygenase (IDO1), a heme-dependent enzyme thatcatalyzes the first and rate-limiting step in tryptophan catabolism toN-formyl-kynurenine, which is then deformylated to generate kynurenine.During infection, the expression of IDO1 is induced by interferon gammato locally deplete tryptophan, which inhibits the growth oftryptophan-dependent intracellular pathogens such as Chlamydiatrachomatis, Toxoplasma gondii, and viruses. Additionally, IDO1 plays arole in preventing oxidative damage in cells, several neuropathologies,regulation of the immune system, and cancer. Although IDO1 activity is acritical component of the immune response to pathogens, prolongedactivity results in the depletion of extracellular tryptophan with theconcomitant production of kynurenine, both of which areimmunosuppressive. IDO1 expression in cancer is well documented andoccurs through both intrinsic activation of IDO1 gene expression and/orthrough the activation of the IFN-γ-to-IDO1 axis, a result of immunecell activation. Additionally, innate immune cells such as dendriticcells, monocytes and macrophages, which are recruited to sites ofinflammation and the tumor microenvironment, are immunosuppressive whenthey express IDO1. Together the IDO1-dependent depletion of tryptophanand production of kynurenine have been linked to suppression of T-cellactivation and proliferation and NK cell function. Furthermore depletionof tryptophan and production of kynurenine are critical for theformation of regulatory T cells (Treg) and myeloid-derived suppressorcells (MDSCs), which function to dampen immune cell activation. TheseIDO1 dependent immunosuppressive mechanisms are components that allowtumors to circumvent the immune system.

Potential inhibitors of kynurenine production through IDO1 inhibitionare already known in the literature. See for example, WO2010005958,WO2012142237 and WO2014150646 and Journal of Medicinal Chemistry (2016),59(1), 419-430. Certain 2,3-dihydro-1H-indole compounds are known in theart. See for example, CAS registry numbers 1359420-19-1, 1358912-57-8,1358648-68-6, 1357815-05-4, 1359035-89-4, and 1359002-77-9.

There is a need for new cancer treatments. In particular there is a needfor new cancer treatments for melanoma, acute myeloid leukemia, chroniclymphocytic leukemia, colorectal cancer, renal cell carcinoma, breastcancer, lung cancer, ovarian cancer, fallopian tube carcinoma, primaryperitoneal carcinoma, cervical cancer, gastric cancer, liver cancer,pancreatic cancer, thyroid cancer, glioma, non-Hodgkin's lymphoma, andHodgkin's lymphoma. There remains a need to provide alternativekynurenine production inhibitors useful in the treatment of cancer.Preferably such compounds have properties that enable optimal dosingrequired for maximal inhibition of tumor cell growth while havingacceptable tolerability for the patient. Preferably such compounds wouldalso be orally bioavailable. Preferably such compounds would also havethe ability to cross the blood brain barrier and thus have brainexposure. Preferably such compounds would also have the ability topotentially overcome resistance to existing kynurenine inhibitors byhaving an alternate mechanism of action.

The present invention provides certain novel 2,3-dihydro-1H-indolecompounds that are inhibitors of kynurenine production. The skilledperson will appreciate that inhibitors of kynurenine production may haveclinical utility as a single agent or in combination with otheranti-cancer agents for the treatment of different types of cancers andin particular melanoma, acute myeloid leukemia, chronic lymphocyticleukemia, colorectal cancer, renal cell carcinoma, breast cancer, lungcancer, ovarian cancer, fallopian tube carcinoma, primary peritonealcarcinoma, cervical cancer, gastric cancer, liver cancer, pancreaticcancer, thyroid cancer, glioma, non-Hodgkin's lymphoma, and Hodgkin'slymphoma.

The present invention also provides a compound of the formula:

wherein:

R1a is hydrogen, methyl, ethenyl, cyano, fluoro, chloro, fluoromethyl,or difluoromethyl;

R1b is hydrogen, fluoro, or chloro;

R1c is hydrogen, hydroxy, fluoro, benzyloxy, or hydroxyethylamino;

R2 is hydrogen or methyl;

R2a is hydrogen or methyl; and

R3a is tetrahydropyranyl.

The present invention provides a compound of the formula:

The present invention also provides a compound of the formula:

The present invention also provides a compound of the formula:

The present invention also provides a compound which is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide.Preferably the compound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamidein a crystalline form. Preferably the compound is crystalline4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamidecharacterized by an X-ray powder diffraction pattern (Cu radiation,λ-1.54060 {right arrow over (Å)}) comprising at least one peak at 17.38°in combination with one or more peaks selected from the group consistingof 12.510, 15.650, 16.37°, 17.56°, 21.48° and 25.23° (2θ±0.2°).

The present invention also provides an intermediate or salt thereof ofthe formula:

useful in the method of making certain compounds of the presentinvention.

The present invention also provides an intermediate or salt thereof ofthe formula:

useful in the method of making certain compounds of the presentinvention.

The present invention also provides an intermediate or salt thereof ofthe formula:

useful in the method of making certain compounds of the presentinvention.

The present invention also provides an intermediate of the formula:

useful in the method of making certain compounds of the presentinvention.

The present invention also provides an intermediate of the formula:

useful in the method of making certain compounds of the presentinvention.

The present invention also provides an intermediate of the formula:

useful in the method of making certain compounds of the presentinvention.

The present invention also provides a pharmaceutical compositioncomprising a compound of the present invention with a pharmaceuticallyacceptable excipient, carrier, or diluent. Preferably the compound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide.

The present invention provides a method of treating a patient with acancer selected from the group consisting of melanoma, acute myeloidleukemia, chronic lymphocytic leukemia, colorectal cancer, renal cellcarcinoma, breast cancer, lung cancer, ovarian cancer, fallopian tubecarcinoma, primary peritoneal carcinoma, cervical cancer, gastriccancer, liver cancer, pancreatic cancer, thyroid cancer, glioma,non-Hodgkin's lymphoma, and Hodgkin's lymphoma comprising administeringto the patient an effective amount of a compound of the presentinvention. Preferably the cancer is melanoma. Preferably the cancer iscolorectal cancer. Preferably the cancer is renal cell carcinoma.Preferably the cancer is breast cancer. Preferably the cancer is lungcancer, in particular non-small cell lung cancer. Preferably the canceris ovarian cancer. Preferably the cancer is glioma. Preferably thecompound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide.

This invention also provides a compound of the present invention for usein therapy. Additionally, this invention provides a compound of thepresent invention for use in the treatment of a cancer selected from thegroup consisting of melanoma, acute myeloid leukemia, chroniclymphocytic leukemia, colorectal cancer, renal cell carcinoma, breastcancer, lung cancer, ovarian cancer, fallopian tube carcinoma, primaryperitoneal carcinoma, cervical cancer, gastric cancer, liver cancer,pancreatic cancer, thyroid cancer, glioma, non-Hodgkin's lymphoma, andHodgkin's lymphoma. Preferably the cancer is melanoma. Preferably thecancer is colorectal cancer. Preferably the cancer is renal cellcarcinoma, Preferably the cancer is breast cancer. Preferably the canceris lung cancer, in particular non-small cell lung cancer. Preferably thecancer is ovarian cancer. Preferably the cancer is glioma. Preferablythe compound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide.Preferably the compound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide.

This invention also provides a combination comprising a compound of thepresent invention and LY3300054 for simultaneous, separate, orsequential use in the treatment of a cancer selected from the groupconsisting of non-small cell lung cancer and colon cancer. Preferablythe cancer is non-small cell lung cancer. Preferably the cancer is coloncancer. Preferably the compound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide.Preferably the compound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamideand the cancer is non-small cell lung cancer. Preferably the compound is4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamideand the cancer is colon cancer.

The following paragraphs describe preferred classes of Formula I:

a) R1a is hydrogen, methyl, cyano, fluoro, or chloro;

b) R1b is hydrogen, fluoro, or chloro;

c) R1c is hydrogen or hydroxy;

d) R2 is hydrogen or methyl;

e) R2a is hydrogen or methyl;

f) R3a is tetrahydropyranyl; and

g) R1a is fluoro, R1b is hydrogen, R1c is hydrogen, R2 is hydrogen, R2ais hydrogen, and R3 is tetrahydropyranyl.

Certain of the compounds of the present invention are crystalline. It iswell known in the crystallography art that, for any given crystal form,the relative intensities of the diffraction peaks may vary due topreferred orientation resulting from factors such as crystal morphologyand habit. Where the effects of preferred orientation are present, peakintensities are altered, but the characteristic peak positions of thepolymorph are unchanged. See, e.g. The U. S. Pharmacopeia 38—NationalFormulary 35 Chapter <941> Characterization of crystalline and partiallycrystalline solids by X-ray powder diffraction (XRPD) Official May 1,2015. Furthermore, it is also well known in the crystallography art thatfor any given crystal form the angular peak positions may vary slightly.For example, peak positions can shift due to a variation in thetemperature or humidity at which a sample is analyzed, sampledisplacement, or the presence or absence of an internal standard. In thepresent case, a peak position variability of ±0.2 in 2θ will take intoaccount these potential variations without hindering the unequivocalidentification of the indicated crystal form. Confirmation of a crystalform may be made based on any unique combination of distinguishing peaks(in units of ° 2θ), typically the more prominent peaks. The crystal formdiffraction patterns, collected at ambient temperature and relativehumidity, were adjusted based on NIST 675 standard peaks at 8.85 and26.77 degrees 2-theta.

As used herein, “treat”, “treating” or “treatment” refers torestraining, slowing, stopping, or reversing the progression or severityof an existing symptom or disorder.

As used herein, the term “patient” refers to a warm blooded animal suchas a mammal, in particular a human, which is afflicted with a particulardisease, disorder, or condition.

One of ordinary skill in the art will appreciate that compounds andcertain intermediates of the invention can exist in tautomeric forms.When any reference in this application to one of the specific tautomersof the compounds of the invention is given, it is understood toencompass both tautomeric forms and all mixtures thereof.

Some intermediates or compounds of the present invention disclosedherein may have one or more chiral or stereogenic centers. Allindividual stereoisomers, enantiomers and diastereomers, as well asmixtures of the enantiomers and diastereomers of all of theseaforementioned compounds or intermediates of the present invention arecontemplated including racemates. It is preferred that compounds orintermediates of the present invention disclosed herein containing atleast one chiral center exist as single enantiomers or diastereomers.The single enantiomer or diastereomer may be prepared beginning withchiral reagents or by stereoselective or stereospecific synthetictechniques (as illustrated in the preparations and examples).Alternatively, the single enantiomer or diastereomers may be isolatedfrom mixtures by standard chiral chromatographic (as illustrated in thepreparations and examples) or crystallization techniques. The skilledartisan will appreciate that in some circumstances the elution order ofenantiomers or diastereomers may be different due to differentchromatographic columns and mobile phases.

The designation of “Isomer 1” in a compound name represents that thecorresponding intermediate or compound of the present invention is thefirst of two eluting enantiomers when a mixture of a pair of enantiomersis separated by chiral chromatography. The designation of “Isomer 2” ina compound name represents that the corresponding intermediate orcompound of the present invention is the second of two elutingenantiomers when the mixture of a pair of enantiomers is separated bychiral chromatography.

The designation of “Isomer A” in a compound name represents that thecorresponding intermediate or compound of the present invention is asingle isomer from a chiral synthesis of unknown absolute configuration.

As used herein, “LY3300054” is an antibody that binds human PD-L (SEQ IDNO: 1), comprising a light chain (LC) and a heavy chain (HC), whereinthe light chain comprises a light chain variable region (LCVR) and theheavy chain comprises a heavy chain variable region (HCVR), and whereinthe LCVR comprises light chain complementarity determining regionsLCDR1, LCDR2, and LCDR3, where the amino acid sequence of LCDR1 isSGSSSNIGSNTVN (SEQ ID NO: 5), the amino acid sequence of LCDR2 isYGNSNRPS (SEQ ID NO: 6), and the amino acid sequence of LCDR3 isQSYDSSLSGSV (SEQ ID NO: 7), and wherein the HCVR comprises heavy chaincomplementarity determining regions HCDR1, HCDR2, and HCDR3, where theamino acid sequence of HCDR1 is KASGGTFSSYAIS (SEQ ID NO: 2), the aminoacid sequence of HCDR2 is GIIPIFGTANYAQKFQG (SEQ ID NO: 3), and theamino acid sequence of HCDR3 is ARSPDYSPYYYYGMDV (SEQ ID NO: 4),respectively.

In some embodiments of LY3300054, the LY3300054 binds to human PD-L1,and comprises a light chain (LC) and a heavy chain (HC), wherein thelight chain comprises a light chain variable region (LCVR) and the heavychain comprises a heavy chain variable region (HCVR), wherein the aminoacid sequence of the LCVR is SEQ ID NO: 9, and the amino acid sequenceof the HCVR is SEQ ID NO: 8. In some embodiments of LY3300054, theLY3300054 binds to human PD-L, comprising a light chain (LC) and a heavychain (HC), wherein the amino acid sequence of the LC is SEQ ID NO: 10and the HC has the amino acid sequence given in SEQ ID NO: 11. In anembodiments of LY3300054, the LY3300054, comprises two light chains andtwo heavy chains, wherein each light chain has the amino acid sequencegiven in SEQ ID NO: 11, and each heavy chain has the amino acid sequencegiven in SEQ ID NO: 10.

As used herein, the term “light chain variable region” or “LCVR” means aportion of a light chain of an antibody molecule that includes aminoacid sequences of CDRs and FRs.

As used herein, the term “heavy chain variable region” “HCVR” means aportion of a heavy chain of an antibody molecule that includes aminoacid sequences of CDRs and FRs.

As used herein, the terms “complementarity determining region” and“CDR”, mean the non-contiguous antigen combining sites found within thevariable region of LC and HC polypeptides of an antibody or anantigen-binding fragment thereof. These particular regions have beendescribed by others including Kabat, et al., Ann. NY Acad. Sci.190:382-93 (1971); Kabat et al., J. Biol. Chem. 252:6609-6616 (1977);Kabat, et al., Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242 (1991); Chothia, et al., J. Mol. Biol. 196:901-917 (1987);MacCallum, et al., J. Mol. Biol., 262:732-745 (1996); and North, et al.,J. Mol. Biol., 406, 228-256 (2011), where the definitions includeoverlapping or subsets of amino acid residue when compared against eachother.

The CDRs are interspersed with regions that are more conserved, termedframework regions (“FR”). Each LCVR and HCVR is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order. FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs ofthe light chain are referred to as “LCDR1, LCDR2, and LCDR3” and thethree CDRs of the heavy chain are referred to as “HCDR1, HCDR2, andHCDR3.” The CDRs contain most of the residues which form specificinteractions with the antigen. The numbering and positioning of CDRamino acid residues within the LCVR and HCVR regions is in accordancewith known conventions (e.g., Kabat (1991), Chothia (1987), and/or North(2011)). In different embodiments of the invention, the FRs of theLY3300054 may be identical to the human germline sequences, or may benaturally or artificially modified.

As used herein, the term “K_(D)” means the equilibrium dissociationconstant of a particular antibody-antigen or antibody fragment-antigeninteraction.

As used herein, the term “binds” means the affinity of an antibody forhuman PD-L1 is intended to mean, unless indicated otherwise, a K_(D) ofless than about 1×10⁻⁶ M, preferably, less than about 1×10⁻⁹ M asdetermined by common methods known in the art, including by use of asurface plasmon resonance (SPR) biosensor at 37° C. essentially asdescribed herein.

As used herein, the following terms have the meanings indicated: “ACN”refers to acetonitrile; “APCI” refers to atmospheric pressure chemicalionization; “BTI” refers to [bis(trifluoroacetoxy)iodo]benzene; “CDI”refers to carbonyldiimidazole; “DCM” refers to dichloromethane; “DMSO”refers to dimethyl sulfoxide; “DMF” refers to N,N-dimethylformamide;“DPBS” refers to Dulbecco's phosphate-buffered saline; “ES” refers toelectrospray ionization; “EtOAc” refers to ethyl acetate; “EtOH” refersto ethanol; “FBS” refers to fetal bovine serum; “HPLC” refers to highperformance liquid chromatography; “iPrOH” refers to isopropanol;“LC/MS-MS” refers to liquid chromatography tandem mass spectrometry;“L-kynurenine-d4” refers to(2S)-2-amino-4-(2-amino-3,4,5,6-tetradeuterio-phenyl)-4-oxo-butanoicacid; “MES” refers to 2-(N-morpholino)ethanesulfonic acid; “MS” refersto mass spectroscopy; “MeOH” refers to methanol; “PBS” refers tophosphate-buffered saline; “TFA” refers to trifluoroacetic acid; “TEA”refers to triethylamine; “THF” refers to tetrahydrofuran; “SFC” refersto supercritical fluid chromatography; and “UVW” refers to ultra-violetwavelength.

The compounds of the present invention can be prepared according to thefollowing schemes, preparations and examples by methods well known andappreciated in the art. Suitable reaction conditions for the steps ofthese preparations and examples are well known in the art andappropriate substitutions of solvents and co-reagents are within theskill of the art. Likewise, it will be appreciated by those skilled inthe art that synthetic intermediates may be isolated and/or purified byvarious well known techniques as needed or desired, and that frequently,it will be possible to use various intermediates directly in subsequentsynthetic steps with little or no purification. Furthermore, the skilledartisan will appreciate that in some circumstances, the order in whichmoieties are introduced is not critical. The particular order of stepsrequired to produce the compounds of the present invention is dependentupon the particular compound being synthesized, the starting compound,and the relative liability of the substituted moieties, as is wellappreciated by the skilled chemist. All substituents, unless otherwiseindicated, are as previously defined, and all reagents are well knownand appreciated in the art.

Compounds of the present invention may be synthesized as illustrated inthe following schemes, where R1a, R1b, R1c, R2, R2a and R3a are aspreviously defined.

Scheme 1 illustrates the general synthesis of compounds of Formula I, R2is H. In Step 1, a 2,3-dihydro-1H-indole (Compound 1) is reacted with anappropriate activated carboxylic acid such as an acid chloride in thepresence of a suitable base such as TEA and in a suitable solvent suchas DCM or dichloroethane (DCE) at an appropriate temperature such as 0°C. to refluxing. A skilled artisan will appreciate that there are manyactivated carboxylic acids and many methods to activate carboxylic acidsin situ to accomplish the reaction of Step 1. The resulting ketone(Compound 2) of Step 1 is then treated with hydroxylamine in a polarprotic solvent such as EtOH at an appropriate temperature such as roomtemperature to refluxing to give the oxime as a mixture of E and Zisomers (Compound 3). Step 3 shows the reduction of the oxime (Compound3) to the amine (Compound 4). The skilled artisan will appreciate thatthere are many methods available to affect this transformation. Forexample, the oxime (Compound 3) is contacted with an appropriatecatalyst such as RANEY® nickel in an appropriate solvent such as MeOH orEtOH in an appropriate reactor such as a PARR® shaker. The mixture isthen subjected to hydrogen pressure such as 100-500 kPa at anappropriate temperature such as room temperature to 50° C. for anappropriate time such as one to 24 hours. Scheme 1, Step 4 depicts theamide coupling of the amine (Compound 4) with an appropriate activatedcarboxylic acid such as an acid chloride in the presence of a suitablebase such as TEA and in a suitable solvent such as DCM or DCE at anappropriate temperature such as 0° C. to refluxing to give a compound ofFormula I. A skilled artisan will appreciate that there are manyactivated carboxylic acids and many methods to activate carboxylic acidsin situ to accomplish the reaction of Step 4. The skilled artisan willfurther appreciate that Scheme 1, Step 3 and Step 4 result in productswith chiral centers. Individual enantiomers may be separated or resolvedby one of ordinary skill in the art at any convenient point in thesynthesis of compounds of the present invention by methods such asselective crystallization techniques or chiral chromatography (See forexample, J. Jacques, et al., “Enantiomers, Racemates, and Resolutions”,John Wiley and Sons, Inc., 1981, and E. L. Eliel and S. H. Wilen,“Stereochemistry of Organic Compounds”, Wiley-Interscience, 1994).

Scheme 2 illustrates an alternate general synthesis of compounds ofFormula I, R2 is H. In Step 1, the ketone (Compound 2) is reacted withan appropriate chiral sulfinamide in the presence of an appropriateLewis acid such as titanium(IV) ethoxide in an appropriate solvent suchas THF at an appropriate temperature such as room temperature to refluxfor an appropriate time such as one to 24 hours to give a chiralethylidenesulfinamide (Compound 5). A skilled artisan will appreciatethat reagents are available to generate either enantiomer of thesulfinamide (Compound 5). Chiral reduction is depicted in Step 2 togenerate an ethylsulfinamide (Compound 6) from an ethylidinesulfinamide(Compound 5) and asterisks are used to indicate chiral centers forclarity. For example, an appropriate catalyst is pre-formed by mixing anappropriate ruthenium reagent such as dichloro(p-cymene)ruthenium(II)dimer with an appropriate aminoethanol such as2-amino-2-methyl-1-propanol in an appropriate solvent such as iPrOH inthe presence of a water scavenger such as 4 Å molecular sieves at anappropriate temperature such as room temperature to refluxing for anappropriate time such as five minutes to approximately one hour. Thepreformed catalyst reaction is cooled to an appropriate temperature suchas room temperature to 50° C. and treated with an ethylidenesulfinamide(Compound 5) and an appropriate base such as potassium tert-butoxide.The reaction is maintained at an appropriate temperature such as roomtemperature to 50° C. for an appropriate time such as one to 24 hours.The skilled artisan will appreciate that there are many catalytic andstoichiometric methods that will affect the same transformation and thatthese methods can result in diastereomeric enrichment depending on thenature of the substrates and the reagents used, up to and includinggeneration of a single diastereomer. Acid hydrolysis of aethylsulfinamide (Compound 6) can be affected by treatment with anappropriate acid such as hydrochloric acid (HCl) in an appropriatesolvent such as dioxane, iPrOH, EtOAc or MeOH at an appropriatetemperature such as 0° C. to room temperature for an appropriate timesuch as one to eight hours to give an amine (Compound 4). The skilledartisan will appreciate that many methods for isolation are known andthese can result in isolation of either the salt or free base of theamine (Compound 4). Step 4 depicts the amide coupling of an amine(Compound 4) with an appropriate activated carboxylic acid analogous toScheme 1, Step 4 above to give a compound of Formula I.

Scheme 3 depicts an alternate general synthesis of a compound of FormulaI, R2 is H. Step 1 depicts an amide coupling of a carboxylic acid(Compound 7) with N,O-dimethylhydroxylamine hydrochloride to give aWeinreb amide (Compound 8). The skilled artisan will appreciate thatthere are many methods to affect this transformation. For example, acarboxylic acid (Compound 7) can be treated with an appropriate couplingreagent such as1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexafluorophosphate (HATU) in the presence of an appropriate base suchas N,N-diisopropylethylamine in an appropriate solvent such as DMF foran appropriate time such as five to ten minutes. The mixture is thentreated with N,O-dimethylhyrdoxylamine hydrochloride and the mixture isstirred at an appropriate temperature such as room temperature to 100°C. for an appropriate time such as three to 18 hours. The resultingWeinreb amide (Compound 8) is then treated with an appropriate Grignard,alkyl lithium or alkyl zinc reagent, in Step 2, to give a ketone(Compound 9). The skilled artisan will appreciate that there are a largenumber of methods available to affect this transformation. For example,the Weinreb amide (Compound 8) in an appropriate solvent such as THF atan appropriate temperature such as 0° C. to −78° C. is treated with anappropriate alkyl metal reagent such as ethylmagnesium bromide. Thereaction is continued after the addition for an appropriate time such asone to 18 hours to give a ketone (Compound 9). Steps 3, 4 and 5 ofScheme 3 are presented for clarity. The methods are analogous to thosepresented in Scheme 1, Steps 2, 3 and 4 respectively. One of skill inthe art will appreciate that compound 11 contains a chiral center andthat chiral purification can be performed on compound 11 or the racemicmixture can be carried forward and separation can be performed after anyof the subsequent steps. Step 6 depicts the deprotection of thetert-butoxycarbonyl protecting group of a carbamate (Compound 12) togive an amine (Compound 13). A skilled artisan will appreciate that thistransformation can be conducted under acid, base or thermal conditions.For example, a carbamate (Compound 12) is contacted with an appropriateacid such as HCl in an appropriate solvent such as dioxane or DCM or amixture thereof at an appropriate temperature such as 0° C. to refluxingfor an appropriate time such as one to 18 hours to give an amine(Compound 13). The skilled artisan will appreciate that there aremethods to isolate an amine as a salt or freebase. Step 7 depicts theamide coupling of an amine (Compound 13) and an activated carboxylicacid to give a compound of Formula I. The conditions are analogous tothose presented in Scheme 1, Step 1.

Scheme 4 depicts an alternate general synthesis of a compound of FormulaI. Step 1 depicts the protection of an amine (Compound 11) with a benzylcarbamate protecting group to give a carbamate (Compound 14). A skilledartisan will appreciate that there are many amine protecting groupsavailable that would be orthogonal protecting groups to thetert-butoxycarbonyl group on compound 14. In an example procedure, anamine (Compound 11) in an appropriate solvent such as DCM and in thepresence of a suitable base such as N,N-diisopropylethylamine iscontacted with benzyloxychloroformate at an appropriate temperature suchas 0° C. to room temperature for an appropriate time such as one to 18hours. The tert-butoxycarbonyl protecting group of Compound 14 isselectively deprotected to give an amine (Compound 15) utilizing methodsanalogous to those described for Scheme 3, Step 6. The resulting amine(Compound 15) is then subjected to an amide coupling reaction with anactivated carboxylic acid by methods analogous to those in Scheme 1,Step 1 to give an amide (Compound 16). Step 4 depicts the deprotectionof the benzyloxy carbamate protecting group of compound 16 to give anamine (Compound 4). A skilled artisan will appreciate that a variety ofmethods are available to affect this transformation. For example,compound 16 is subjected to catalytic hydrogenation with an appropriatecatalyst such as palladium hydroxide in an appropriate solvent such asEtOH under an appropriate hydrogen pressure such as 100 to 500 kPa foran appropriate time such as one to eight hours to give the amine(Compound 4). Finally, the conversion of amine (Compound 4) to acompound of Formula I is as described in Scheme 1, Step 4.

Scheme 5 depicts an alternate general synthesis of a compound of FormulaI. Step 1 depicts the amide coupling of an amine (Compound 17) and anactivated carboxylic acid to give an amide (Compound 18). The conditionsare analogous to those presented in Scheme 1, Step 1. Step 2 depicts theformation of alpha aryl ester (Compound 19) through the catalytic crosscoupling of a bromide (Compound 18) with an ester enolate. The skilledartisan will appreciate the wide range of conditions that can affectthis transformation. For example, a solution of an appropriate dialkylamine such as dicyclohexylamine in an appropriate solvent such astoluene is treated with an appropriate lithium base such asn-butyllithium at an appropriate temperature such as 0° C. to −78° C.for an appropriate time such as 10 minutes to one hour. This solution istreated with a solution of an appropriate ester such as methyl2-methylpropanoate in an appropriate solvent such as toluene and theresulting mixture is stirred for an appropriate time such as 10 minutesto one hour at an appropriate temperature such as 0° C. to −40° C. Theresulting mixture is then treated with an appropriate palladium catalystsuch as di-μ-bromobis(tri-t-butylphosphine)dipalladium(I) and themixture is stirred at an appropriate temperature such as 0° C. to roomtemperature for an appropriate time such as one to 18 hours to give analpha aryl ester (Compound 19). An ester (Compound 19) can be hydrolyzedto an acid (Compound 20) by methods well known in the art. For example,an ester (Compound 19) is contacted with a suitable base such aspotassium trimethylsilanolate in an appropriate solvent such as THF atan appropriate temperature such as room temperature to refluxing for anappropriate time such as one to seven days. Step 4 depicts the amidecoupling of a carboxylic acid (Compound 20) with ammonia to give acarboxamide (Compound 21). The skilled artisan will appreciate the manymethods available to active a carboxylic acid as well as introduceammonia sources. For example, a carboxylic acid (Compound 20) iscontacted with 1,1′-carbonyldiimidazole in an appropriate solvent suchas DCM or DMF or a mixture thereof at an appropriate temperature such as0° C. to refluxing for an appropriate time such as 30 minutes to eighthours. Ammonium hydroxide is added to the mixture and the reaction iscontinued for an additional time such as one to 18 hours. Step 21depicts the Hoffman rearrangement of a carboxamide (Compound 21) to anamine (Compound 4). The skilled artisan will appreciate the wide varietyof reagents and conditions that can affect this transformation. Forexample, a solution of a carboxamide (Compound 21) in an appropriatesolvent such as a mixture of ACN and water is treated with[bis(trifluoroacetoxy)iodo]benzene at appropriate temperature such asroom temperature to refluxing for an appropriate time such as one to 18hours. Finally, the conversion of amine (Compound 4) to a compound ofFormula I is describe in Scheme 1, Step 4.

Preparation 1 Synthesis of1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethanone

Add TEA (51.9 mL, 372.2 mmol) and tetrahydropyran-4-carbonyl chloride(22.1 g, 148.9 mmol) to a mixture of 1-indolin-5-ylethanone (20.0 g,124.1 mmol) in DCM (496 mL). Stir the resulting mixture at roomtemperature for two hours. Dilute the reaction mixture with DCM (500 mL)and wash with saturated aqueous sodium bicarbonate. Isolate the organiclayer and extract the aqueous layer twice with DCM (500 mL). Washcombined organic layers with saturated aqueous sodium chloride, dry overanhydrous sodium sulfate, filter and concentrate the filtrate to givethe title compound quantitatively as a light yellow solid. ES/MS (m/z):274.0 (M+H).

Alternative Isolation Procedure:

Treat the product with heptane and concentrate. Repeat the treatment andconcentration a second time. Treat with heptane and cool to 0-5° C.Collect the product by filtration and rinse with heptane and dry to givethe title compound.

Preparation 2 Synthesis of[5-(N-hydroxyethanimidoyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone

Add hydroxylamine (50 mass % in water, 22.8 mL, 372 mmol) to a mixtureof1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethanone(Preparation 1) (33.9 g, 124.0 mmol) in EtOH (1240 mL). Stir theresulting mixture at room temperature for three days. Concentrate thereaction mixture to give the title compound as a mixture of E/Z isomersquantitatively. ES/MS (m/z): 289.0 (M+H).

Preparation 3 Synthesis of racemic[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone

Add RANEY® nickel (slurry in EtOH, 60 g) to a 2250 mL PARR® shakerbottle and purge with nitrogen. Add 2M ammonia in MeOH (700 mL) and thena solution of[5-(N-hydroxyethanimidoyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone(Preparation 2) (35.8 g, 124.0 mmol) in 2M ammonia in MeOH (700 mL).Cool the potentially exothermic mixture to room temperature ifnecessary, seal, purge with nitrogen and then hydrogen. Stir underhydrogen (60 psi, or 414 kPa) for four hours at room temperature. Filteroff the solids and concentrate the filtrate. Purify by silica gel columnchromatography with 7-26% (7M ammonia in MeOH) in EtOAc to give thetitle compound (26.5 g, 78%) as an off-white solid. ES/MS (m/z): 275.0(M+H).

Preparation 4A and B Separation of[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone,Isomer 1 and[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone,Isomer 2

Purify racemic[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone(Preparation 3) by chiral SFC to afford the first eluting enantiomer(Isomer 1). ES/MS (m/z): 275.0 (M+H). Purification conditions:CHIRALPAK® AD-H, 50×150 cm column; Mobile phase: 40% EtOH (containing0.5% N,N-dimethylethylamine) in CO₂; Column temperature: 40° C.; Flowrate: 300 g/minute; UVW: 260 nm. Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical SFC (>99% ee, R_(t): 1.35 minutes; Column:CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase: 40% EtOH (containing 0.5%N,N-dimethylethylamine) in CO₂; Flow rate: 5 mL/minute; UVW: 260 nm) orby chiral analytical HPLC (97.4% ee, R_(t): 6.48 minutes; Column:CHIRALPAK® AD-H, 4.6 mm×150 mm; Mobile phase: 100% EtOH (containing 0.2%isopropylamine); Flow rate: 1 mL/minute; UVW: 225 nm).

The above purification also yields the second eluting enantiomer (Isomer2). ES/MS (m/z): 275.1 (M+H). Confirm enantiomeric enrichment of Isomer2 by chiral analytical SFC (97.2% ee, R_(t): 1.85 minutes; Column:CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase: 40% EtOH (containing 0.5%N,N-dimethylethylamine) in CO₂; Flow rate: 5 mL/minute; UVW: 260 nm) orby chiral analytical HPLC (97.6% ee, R_(t): 5.31 minutes; Column:CHIRALPAK® AD-H, 4.6 mm×150 mm; Mobile phase: 100% EtOH (containing 0.2%isopropylamine); Flow rate: 1 mL/minute; UVW: 225 nm).

Preparation 5 Synthesis of(R)-2-methyl-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethylidene}propane-2-sulfinamide

Add titanium(IV) ethoxide (5.0 g, 21.9 mmol) to a mixture of1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethanone(Preparation 1) (3.0 g, 11.0 mmol) and (R)-2-methylpropane-2-sulfinamide(1.6 g, 13.2 mmol) in THF (43.9 mL). Reflux the resulting mixture for 24hours. Cool the reaction mixture to room temperature. Dilute the mixturewith EtOAc (100 mL) and saturated aqueous sodium chloride (40 mL) andstir vigorously for 15 minutes. Isolate the organic layer and extractthe aqueous layer twice with EtOAc (100 mL). Wash the combined organiclayers with saturated aqueous sodium chloride, dry over anhydrous sodiumsulfate, filter and concentrate the filtrate. Purify by silica gelcolumn chromatography with 10-100% ACN in DCM to give the title compound(3.7 g, 87%) as a light yellow solid. ES/MS (m/z): 377.0 (M+H).

Alternative Isolation Procedure:

Instead of cooling the reaction to room temperature, cool the reactionmixture to 10° C. and then filter. Rinse the solid with toluene and dryto give the title compound.

Preparation 6 Synthesis of(R)-2-methyl-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}propane-2-sulfinamide,Isomer A

Heat a mixture of dichloro(p-cymene)ruthenium(II) dimer (0.021 g, 0.033mmol), 2-amino-2-methyl-1-propanol (0.006 g, 0.066 mmol) and molecularsieves (4 Å, 0.5 g) in iPrOH (2 mL) to reflux and then cool to 50° C.Add a solution of(R)-2-methyl-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethylidene}propane-2-sulfinamide(Preparation 5) (0.5 g, 1.33 mmol) in iPrOH (8.8 mL) and a solution ofpotassium tert-butoxide (0.019 g, 0.17 mmol) in iPrOH (1.6 mL). Heat theresulting mixture at 55° C. for two hours. Then heat an additionalmixture of dichloro(p-cymene)ruthenium(II) dimer (0.021 g, 0.033 mmol),2-amino-2-methyl-1-propanol (0.06 g 0.66 mmol) and molecular sieves (4Å, 0.5 g) in iPrOH (2 mL) to reflux, cool to 50° C. and add to the abovereaction mixture. Add a solution of potassium tert-butoxide (0.019 g,0.17 mmol) in iPrOH (1.6 mL) to the above reaction mixture. Heat themixture at 55° C. for 20 minutes. Cool the reaction to room temperatureand stir overnight. Dilute the reaction with DCM (20 mL) and filterthrough a diatomateous earth pad. Wash the pad with 5% MeOH in DCM andconcentrate the filtrate to give the title compound quantitatively.ES/MS (m/z): 379.0 (M+H).

Alternative Isolation Procedure:

Instead of cooling the reaction to room temperature, cool the reactionmixture to 28-32° C. and then filter through diatomaceous earth. Rinsethe filtering solid with dichloromethane and concentrate to give thetitle compound.

Preparation 7A and 7B Synthesis of[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone,Isomer 1

Add hydrochloric acid (4 M in 1,4-dioxane, 1.66 mL, 6.64 mmol) to amixture of(R)-2-methyl-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}propane-2-sulfinamide,Isomer A (Preparation 6) (503 mg, 1.33 mmol) in MeOH (6.6 mL). Stir theresulting mixture at room temperature for one hour. Concentrate andpurify the residue by reverse phase chromatography (Redisep Rf Gold HighPerformance C18 Reverse Phase Column, 0-100% ACN in 10 mM aqueousammonium bicarbonate). Concentrate to give the title compound (265 mg,73%). Confirm enantiomeric enrichment by chiral analytical HPLC (98.8%ee, R_(t): 6.40 minutes; Column: CHIRALPAK® AD-H, 4.6 mm×150 mm; Mobilephase: 100% EtOH (containing 0.2% isopropylamine); Flow rate: 1mL/minute; UVW: 225 nm). Confirmed to be preparation 4A, Isomer 1. ES/MS(m/z): 275.0 (M+H).

Synthesis of[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanonehydrochloride, Isomer 1

Add hydrochloric acid (5.5 M in iPrOH, 400 mL, 2.20 mol) to a 5° C.slurry of(R)-2-methyl-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}propane-2-sulfinamide,Isomer A (162.3 g, 364 mmol) in EtOAc (1.2 L) dropwise with overheadmechanical stirring. Remove cooling bath after addition of 100 mL of theacid solution. Continue the addition and stir the resulting mixture atroom temperature for three hours. Cool to 3° C. and filter. Rinse thefilter cake with 1-1.5 L of EtOAc, until washes are clear. Dry thecollected solids in a house vacuum oven at 60° C. to give the titlecompound as an off white solid (96.4 g, 82.5%). Confirm enantiomericenrichment by chiral analytical HPLC (98% ee, R_(t): 6.45 minutes;Column: CHIRALPAK® AD-H, 4.6 mm×150 mm; Mobile phase: 100% EtOH(containing 0.2% isopropylamine); Flow rate: 1 mL/minute; UVW: 225 nm).Confirmed to be preparation 4A, Isomer 1. ES/MS (m/z): 275.0 (M+H).Confirmed to be preparation 4A, Isomer 1. ES/MS (m/z): 275.1 (M+H).

Prepare the following compounds essentially analogous to Preparation 1.

Prep No. Chemical Name Physical data 8[5-Bromo-2,3-dihydro-1H-indol-1-yl] ES/MS (m/z,(tetrahydro-2H-pyran-4-yl)methanone ⁷⁹Br/⁸¹Br): 310.0/312.0 (M + H) 9Benzyl {1-[1-(tetrahydro-2H-pyran-4- ES/MS (m/z): 423.2ylcarbonyl)-2,3-dihydro-1H-indol-5-yl] (M + H) propyl}carbamate, IsomerA

Preparation 10 Synthesis of tert-butyl5-acetyl-2,3-dihydro-1H-indole-1-carboxylate

Treat a 100° C. solution of 1-indolin-5-ylethanone (1.00 g, 6.02 mmol)in toluene (12 mL) with a solution of di-tert-butyl dicarbonate (1.97 g,9.03 mmol) in toluene (12 mL) dropwise over 20 minutes. Continue heatingthe mixture for 30 minutes. Concentrate the reaction mixture. Purify bysilica gel column chromatography with 15-35% (1:1 EtOAc:DCM) in hexanesto give the title compound (1.52 g, 97%) as a white solid. ES/MS (m/z):262.0 (M+H).

Preparation 11 Synthesis of tert-butyl5-[methoxy(methyl)carbamoyl]-2,3-dihydro-1H-indole-1-carboxylate

Treat a solution of1-(tert-butoxycarbonyl)-2,3-dihydro-1H-indole-5-carboxylic acid (14.0 g,53.2 mmol) in DMF (200 mL) and N,N-diisopropylethylamine (28.0 mL, 161mmol) with1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexafluorophosphate (23.3 g, 61.1 mmol). Stir the resulting mixture atroom temperature for five minutes. Add N,O-dimethylhydroxylaminehydrochloride (7.26 g, 74.4 mmol). Stir the resulting mixture overnightat room temperature. Dilute the reaction mixture with EtOAc and washwith saturated aqueous sodium bicarbonate. Isolate the organic layer andextract the aqueous layer twice with EtOAc. Wash the combined organiclayers twice with water, adding saturated aqueous sodium chloride to aidphase separation. Dry the organic layer over anhydrous sodium sulfate,filter and concentrate the filtrate. Purify by silica gel columnchromatography with 10-32% acetone in hexanes to give the title compoundas a clear, colorless, thick oil in quantitative yield. ES/MS (m/z):307.0 (M+H).

Preparation 12 Synthesis of tert-butyl5-propanoyl-2,3-dihydro-1H-indole-1-carboxylate

Cool a solution of tert-butyl5-[methoxy(methyl)carbamoyl]-2,3-dihydro-1H-indole-1-carboxylate(Preparation 11) (14.3 g, 46.7 mmol) in THF (311 mL, anhydrous) to 0° C.Add ethylmagnesium bromide (3M in diethylether, 39.0 mL, 117 mmol)dropwise over 25 minutes. After stirring at 0° C. for 1.5 hours,cautiously quench the reaction mixture with saturated aqueous ammoniumchloride solution. Extract three times with EtOAc. Dry the combinedorganic layers over anhydrous sodium sulfate, filter and concentrate thefiltrate. Purify by silica gel column chromatography with 15-30% (1:1EtOAc:DCM) in hexanes to give the title compound (11.7 g, 91%) as awhite solid. ES/MS (m/z): 276.0 (M+1).

Prepare the following compounds essentially analogous to Preparation 2,except Preparation 14, in which heating at 70° C. increases the reactionrate.

Prep No. Chemical Name Physical data 13 tert-Butyl5-(N-hydroxyethanimidoyl)- ES/MS (m/z): 277.02,3-dihydro-1H-indole-1-carboxylate (M + H) 14 tert-Butyl5-(N-hydroxypropanimidoyl)- ES/MS (m/z): 291.02,3-dihydro-1H-indole-1-carboxylate (M + H)

Prepare the following compounds essentially analogous to Preparation 3.

Prep No. Chemical Name Physical data 15 Racemic tert-butyl5-[1-aminoethyl]- ES/MS (m/z): 246.0 2,3-dihydro-1H-indole-1-carboxylate(M − NH2)⁺ 16 Racemic tert-butyl 5-[1-aminopropyl]- ES/MS (m/z): 260.12,3-dihydro-1H-indole-1-carboxylate (M − NH2)⁺

Preparation 17A and B Separation of tert-butyl5-[1-aminopropyl]-2,3-dihydro-H-indole-1-carboxylate, Isomer 1 andtert-butyl 5-[1-aminopropyl]-2,3-dihydro-1H-indole-1-carboxylate, Isomer2 (for Chiral Separation)

Purify racemic tert-butyl5-[1-aminopropyl]-2,3-dihydro-1H-indole-1-carboxylate (Preparation 16)by chiral chromatography to afford the first eluting enantiomer (Isomer1). MS (m/z): 260.0 (M-NH2)⁺. Purification conditions: CHIRALPAK® AD,8×33.5 cm column; Mobile phase: 100% MeOH (containing 0.2%N,N-dimethylethylamine); Flow rate: 400 mL/minute; UVW: 240 nm. Confirmenantiomeric enrichment of Isomer 1 by chiral analytical HPLC (>99% ee,R_(t): 9.2 minutes; Column: CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase:100% MeOH (containing 0.2% N,N-dimethylethylamine); Flow rate: 0.6mL/minute; UVW: 280 nm).

The above purification also yields the second eluting enantiomer (Isomer2). ES/MS (m/z): 260.0 (M-NH2)⁺. Confirm enantiomeric enrichment ofIsomer 2 by chiral analytical HPLC (99% ee, R_(t): 14.7 minutes; Column:CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase: 100% MeOH (containing 0.2%N,N-dimethylethylamine); Flow rate: 0.6 mL/minute; UVW: 280 nm).

Preparation 18 Synthesis of racemic tert-butyl5-{1-[(4-fluorobenzoyl)amino]ethyl}-2,3-dihydro-1H-indole-1-carboxylate

Treat a solution of racemic tert-butyl5-[1-aminoethyl]-2,3-dihydro-1H-indole-1-carboxylate (Preparation 15)(3.00 g, 11.4 mmol) in DCM (57 mL) with N,N-diisopropylethylamine (4.0mL, 57.2 mmol). Add 4-fluorobenzoyl chloride (1.52 mL, 12.6 mmol) andstir overnight. Quench the reaction mixture with water and add saturatedaqueous sodium bicarbonate solution. Separate the layers and extracttwice with DCM. Dry the combined organic layers over anhydrous sodiumsulfate, filter and concentrate the filtrate. Purify by silica gelcolumn chromatography with 50-75% (10% acetone in DCM) in hexanes togive the title compound (4.29 g, 98%) as a pale yellow solid. ES/MS(m/z): 407.0 (M+Na)⁺, 383.0 (M−H)⁻.

Prepare the following compound essentially analogous to Preparation 18.

Prep No. Chemical Name Physical data 19 Racemic tert-butyl 5-{1-[(4-ES/MS (m/z): 421.0 fluorobenzoyl)amino]propyl}-2,3- (M + Na)⁺, 397.2 (M− H)⁻ dihydro-1H-indole-1-carboxylate

Preparation 20 Synthesis of tert-butyl5-[1-{[(benzyloxy)carbonyl]amino}propyl]-2,3-dihydro-1H-indole-1-carboxylate,Isomer A

Treat a solution of tert-butyl5-[1-aminopropyl]-2,3-dihydro-H-indole-1-carboxylate, Isomer 2(Preparation 17B) (3.25 g, 11.8 mmol) in DCM (47.0 mL) at roomtemperature with N,N-diisopropylethylamine (4.53 mL, 25.9 mmol). Addbenzyl chloroformate (2.12 mL, 14.1 mmol) and stir overnight. Treat thereaction mixture with additional N,N-diisopropylethylamine (2.06 mL,11.8 mmol) and benzyl chloroformate (0.707 mL, 4.70 mmol). Stir for 30minutes. Quench the reaction mixture with water and add saturatedaqueous sodium bicarbonate solution. Separate the layers and extract theaqueous layer twice with DCM. Dry the combined organic layers overanhydrous sodium sulfate, filter and concentrate the filtrate. Purify bysilica gel column chromatography eluting with a gradient of 5-35%acetone in hexanes to give the title compound (4.47 g, 93%) as a whitefoam. ES/MS (m/z): 433.2 (M+Na)⁺, 409.0 (M−H)⁻.

Preparation 21 Synthesis of racemicN-[1-(2,3-dihydro-1H-indol-5-yl)ethyl]-4-fluorobenzamide

Add 4M hydrochloric acid in 1,4-dioxane (27.9 mL, 112 mmol) to a mixtureof racemic tert-butyl5-{1-[(4-fluorobenzoyl)amino]ethyl}-2,3-dihydro-1H-indole-1-carboxylate(Preparation 18) (4.29 g, 11.2 mmol) in DCM (112 mL). Heat the resultingmixture at 40° C. for six hours. Neutralize with 2N aqueous NaOHsolution. Add 4:1 CHCl₃:IPA to dissolve gummy solids and separate thelayers. Wash the organic layer with brine, dry over anhydrous sodiumsulfate, filter and concentrate the filtrate. Purify by silica gelcolumn chromatography eluting with a gradient of 30-60% (25% acetone inDCM) in hexanes to give the title compound (2.48 g, 78%) as a slightlyoff-white foam. ES/MS (m/z): 285.0 (M+H).

Prepare the following compounds essentially analogous to Preparation 21.

Prep No. Chemical Name Physical data 22 RacemicN-[1-(2,3-Dihydro-1H-indol- ES/MS (m/z): 5-yl)propyl]-4-fluorobenzamide299.0 (M + H) 23 Benzyl [1-(2,3-dihydro-1H-indol-5- ES/MS (m/z):yl)propyl]carbamate, Isomer A 311.2 (M + H)

Preparation 24A and B Separation ofN-[1-(2,3-dihydro-1H-indol-5-yl)ethyl]-4-fluorobenzamide, Isomer 1 andN-[1-(2,3-dihydro-1H-indol-5-yl)ethyl]-4-fluorobenzamide, Isomer 2 (forChiral Separation)

Purify racemic N-[1-(2,3-dihydro-1H-indol-5-yl)ethyl]-4-fluorobenzamide(Preparation 21) by chiral SFC to afford the first eluting enantiomer(Isomer 1). ES/MS (m/z): 285.0 (M+H). Purification conditions:CHIRALPAK® AS-H, 5×15 cm column; Mobile phase: 15% iPrOH in CO₂; Columntemperature: 40° C.; Flow rate: 300 g/minute; UVW: 250 nm. Confirmenantiomeric enrichment of Isomer 1 by chiral analytical SFC (>99% ee,R_(t): 1.47 minutes; Column: CHIRALPAK® AS-H, 4.6×150 mm; Mobile phase:25% iPrOH in CO₂; Flow rate: 5 mL/minute; UVW: 300 nm).

The above purification also yields the second eluting enantiomer (Isomer2). ES/MS (m/z): 285.0 (M+H). Confirm enantiomeric enrichment of Isomer2 by chiral analytical SFC (98.5% ee, R_(t): 2.08 minutes; Column:CHIRALPAK® AS-H, 4.6×150 mm; Mobile phase: 25% iPrOH in CO₂; Flow rate:5 mL/minute; UVW 300 nm).

Preparation 25 Synthesis of{5-[1-aminopropyl]-2,3-dihydro-1H-indol-1-yl}(tetrahydro-2H-pyran-4-yl)methanone,Isomer A

Add a solution of benzyl{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propyl}carbamate,Isomer A (Preparation 9) (2.12 g, 5.02 mmol) in EtOH (35 mL) to anitrogen purged suspension of 20% palladium hydroxide on carbon (2.12 g)in EtOH (35 mL) in a PARR® shaker bottle. Seal, purge with nitrogen andthen hydrogen. Shake under an atmosphere of hydrogen at 414 kPa (60 psi)for 3.6 hours at room temperature. Filter the reaction mixture throughdiatomaceous earth and concentrate the filtrate to give the titlecompound (1.36 g, 94%) as a grayish-white solid. ES/MS (m/z): 289.2(M+H), 272.0 (M-NH2)⁺.

Preparation 26 Synthesis of methyl2-methyl-2-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propanoate

Add n-butyllithium (3.6 mL, 8.9 mmol, 2.2 M in hexanes) to a stirred 0°C. solution of dicyclohexylamine (1.9 mL, 9.6 mmol) in toluene (25 mL)under nitrogen and stir for 20 minutes. Add a solution of methylisobutyrate (0.83 g, 8.2 mmol) in toluene (5 mL) dropwise to thepreviously prepared mixture and stir for 30 minutes at 0° C. Add[5-bromo-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone(Preparation 8) (2.3 g, 7.4 mmol) and degas the resulting mixture withnitrogen. Add di-μ-bromobis(tri-t-butylphosphine)dipalladium(I) (50 mg,0.06 mmol) and allow the resulting mixture to warm to room temperatureunder nitrogen. After two hours, add a second portion ofdi-μ-bromobis(tri-t-butylphosphine)dipalladium(I) (50 mg, 0.06 mmol) andstir at room temperature under nitrogen overnight. Dilute with EtOAc andaqueous 1N HCl solution and stir for 10 minutes. Filter and rinse thesolids with EtOAc. Separate the filtrate layers and wash the organiclayer with saturated aqueous sodium bicarbonate solution and brine. Drythe organic layer over anhydrous sodium sulfate, filter and concentratethe filtrate. Purify by silica gel column chromatography with 20-60%EtOAc in hexanes to give the title compound (2.3 g, 94% yield, 89%purity) as a white solid. ES/MS (m/z): 332.2 (M+H).

Preparation 27 Synthesis of2-methyl-2-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propanoicAcid

Dissolve methyl2-methyl-2-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propanoate(Preparation 26) (2.2 g, 6.6 mmol) in THF (66 mL) and add potassiumtrimethylsilanolate (1.1 g, 8.6 mmol). Stir at room temperature for fourdays. Filter the solids and wash with THF. Dissolve the solids in waterand acidify with aqueous 5N HCl solution. Cool in a refrigerator for 30minutes. Filter to collect the title compound (1.20 g, 57%) as a whitesolid. ES/MS (m/z): 318.0 (M+H).

Preparation 28 Synthesis of2-methyl-2-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propanamide

Add 1,1′-carbonyldiimidazole (126 mg, 0.763 mmol) to a stirred solutionof2-methyl-2-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propanoicacid (Preparation 27) (202 mg, 0.636 mmol) in DCM (6.4 mL) and stir atroom temperature under nitrogen for 45 minutes. Add ammonium hydroxide(1 mL, 9.55 mmol, 25% w/v in water) and stir at room temperature undernitrogen for 90 minutes. Add DMF (3 mL) to aid solubility and continuestirring at room temperature for two hours. Concentrate and purify bysilica gel column chromatography with 0-10% EtOH in DCM to obtain thetitle compound (180 mg, 89%) as a white solid. ES/MS (m/z): 317.0 (M+H).

Preparation 29 Synthesis of[5-(1-amino-1-methylethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydropyran-4-yl)methanone

Add [bis(trifluoroacetoxy)iodo]benzene (115 mg, 0.26 mmol) to a stirredmixture of2-methyl-2-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propanamide(Preparation 28) (81 mg, 0.26 mmol) in ACN (0.25 mL) and water (0.25 mL)and stir at room temperature under nitrogen overnight. Concentrate toobtain the title compound (140 mg, 94%, 50% pure material) as a lightpink solid. ES/MS (m/z): 289.2 (M+H), 272.0 (M-NH2)⁺.

Preparation 30 Synthesis of methyl4-fluoro-2-[(2-hydroxyethyl)amino]benzoate

Heat a mixture of methyl-2-amino-4-fluorobenzoate (2.81 g, 15.9 mmol)and 2-iodoethanol (0.879 mL, 11.2 mmol) to 90° C. for six hours thencool to room temperature. Dissolve the neat mixture in EtOAc, wash threetimes with aqueous 1N NaOH solution, followed by brine. Dry the organiclayer over anhydrous magnesium sulfate, filter and concentrate thefiltrate to obtain 2.94 g of light brown oil. Add 2-iodoethanol (1.26mL, 15.9 mmol) and heat the mixture at 100° C. overnight. Add additional2-iodoethanol (0.314 mL, 3.99 mmol) and continue heating at 100° C. fortwo hours. Cool to room temperature. Dissolve the neat mixture in EtOAc,wash three times with aqueous 1N NaOH solution, followed by brine. Drythe organic layer over anhydrous magnesium sulfate, filter andconcentrate the filtrate to obtain 2.50 g brown solids. Purify by silicagel column chromatography with 20-40% EtOAc in hexanes to give the titlecompound (1.12 g, 33%) as a white solid. ES/MS (m/z): 214.0 (M+H).

Preparation 31 Synthesis of 4-fluoro-2-[(2-hydroxyethyl)amino]benzoicAcid

Add sodium hydroxide (0.49 mL, 2.4 mmol, 5M in water) to a stirredsolution of methyl 4-fluoro-2-[(2-hydroxyethyl)amino]benzoate(Preparation 30) (104 mg, 0.488 mmol) in 1,4-dioxane (2.4 mL). Stircapped at room temperature for 30 minutes then heat to 70° C. for twohours. Concentrate and acidify to approximately pH 1-2 with aqueous 1 NHCl, extract twice with DCM. Dry combined organic layer over anhydrousmagnesium sulfate, filter and concentrate the filtrate to obtain thetitle compound (89 mg, 92%) as tan solids. ES/MS (m/z): 200.0 (M+H).

Reference Preparation 1 Synthesis of1-(2,4-difluorophenyl)-3-(2,3-dihydro-1H-indol-5-ylmethyl)urea

Stir a mixture of tert-butyl 5-(aminomethyl)indoline-1-carboxylate (4.1g, 17 mmol) and 2,4-difluoro-1-isocyanatobenzene (3 mL, 24 mmol) in DCM(100 mL) for one hour. Quench the reaction with MeOH and water andconcentrate. Dissolve the residue in DCM (30 mL) and add TFA (15 mL) andallow to stand at room temperature for two hours. Concentrate and addsaturated aqueous sodium bicarbonate. Extract the mixture with MeOH/DCM(1/5, v/v). Dry the organic layer over anhydrous magnesium sulfate,filter and concentrate the filtrate. Recrystallize from EtOH to give twocrops. Combine the crops to give the title compound (3.5 g, 68%). ES/MS(m/z): 304.0 (M+H).

Reference Preparation 2 Synthesis of1-(2,4-difluorophenyl)-3-{[1-(3,4,5-tribromobenzoyl)-2,3-dihydro-1H-indol-5-yl]methyl}urea

Degas (N₂) a solution of1-(2,4-difluorophenyl)-3-(2,3-dihydro-1H-indol-5-ylmethyl)urea(Reference Preparation 1) (70 mg, 0.23 mmol), 3,4,5-tribromobenzoic acid(170 mg, 0.24 mmol) and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (134 mg, 0.35 mmol) in DMF (2 mL). Add TEA(0.08 mL, 0.6 mmol) and stir at room temperature for one hour. Directlypurify the reaction mixture by reverse phase purification (Column:Redisep Rf Gold High Performance C18 Reverse Phase Column; eluent: A: 10mM ammonium bicarbonate in water with 5% MeOH (pH 10), B: ACN; gradient:40% B for 5 minutes then 40-95% B over 15 minutes; flow 60 mL/minute,UVW 219/254 nM) and isolate the product by lyophilization to give thetitle compound (149 mg, 54%). ES/MS (m/z, ⁷⁹Br/⁸¹Br): 644.0/646.0 (M+H).

Example 1 Racemic4-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

Combine racemic[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone(Preparation 3) (420 mg, 1.53 mmol) and 4-fluorobenzoic acid (257 mg.1.84 mmol) in DCM (15 mL). To the stirring solution addN,N-diisopropylethylamine (534 μL, 3.06 mmol) and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexafluorophosphate (890 mg, 2.30 mmol). Stir the resulting mixture atroom temperature for 16 hours. Evaporate the solvent and purify byreverse phase column chromatography (Redisep Rf Gold High PerformanceC18 Reverse Phase Column, 25-100% ACN in 10 mM aqueous ammoniumbicarbonate) to give the title compound (372 mg, 61%). ES/MS (m/z):397.2 (M+H). ¹H NMR (d₆-DMSO) δ 8.73 (d, J=8 Hz, 1H), 7.98 (d, J=8 Hz,1H), 7.93-7.89 (m, 2H), 7.28-7.23 (m, 2H), 7.22 (s, 1H), 7.12 (d, J=8Hz, 1H), 5.07 (quin, J=8 Hz, 1H), 4.14 (t, J=8 Hz, 2H), 3.85 (m, 2H),3.36 (m, 2H), 3.09 (t, J=8 Hz, 2H), 2.80 (m, 1H), 1.57-1.66 (m, 4H),1.41 (d, J=7 Hz, 3H).

Example 1A4-Fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

Synthetic Method 1:

Purify racemic4-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide(Example 1) by chiral SFC to afford the first eluting enantiomer as thetitle compound. ES/MS (m/z): 397.0 (M+H). Purification conditions:CHIRALPAK® AD-H, 21×150 mm; Mobile phase: 40% MeOH in CO₂; Columntemperature: 40° C.; Flow rate: 70 g/minute; UVW: 225 nm. Confirmenantiomeric enrichment of Isomer 1 by chiral analytical SFC (>99% ee,R_(t): 1.72 minutes; Column: CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase:40% MeOH in CO₂; Flow rate: 5 mL/minute; UVW: 225 nm). ¹H NMR (d₆-DMSO)δ 8.73 (d, J=8 Hz, 1H), 7.98 (d, J=8 Hz, 1H), 7.93-7.89 (m, 2H),7.28-7.23 (m, 2H), 7.22 (s, 1H), 7.12 (d, J=8 Hz, 1H), 5.07 (quin, J=8Hz, 1H), 4.14 (t, J=8 Hz, 2H), 3.85 (m, 2H), 3.36 (m, 2H), 3.09 (t, J=8Hz, 2H), 2.80 (m, 1H), 1.57-1.66 (m, 4H), 1.41 (d, J=7 Hz, 3H).

Synthetic Method 2:

Add TEA (9.8 mL, 70.3 mmol) and then 4-fluorobenzoyl chloride (5.85 g,36.9 mmol) to a solution of[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanone,Isomer 1 (Preparation 4A) (9.65 g, 35.2 mmol) in DCM (176 mL) at 0° C.Allow the resulting mixture to warm to room temperature and stirovernight. Dilute the reaction mixture with EtOAc (300 mL), filterthrough a silica gel pad and wash with EtOAc. Concentrate the filtrateand purify by silica gel column chromatography with a gradient from25-30% ACN in DCM to give the title compound (9.4 g, 67.1%) as anoff-white solid. MS (m/z): 397.2 (M+H). Confirm enantiomeric enrichmentby chiral analytical SFC (>99% ee, R_(t): 1.74 minutes; Column:CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase: 40% MeOH in CO₂; Flow rate: 5mL/minute; UVW: 225 nm). ¹H NMR (d₆-DMSO) δ 8.73 (d, J=8 Hz, 1H), 7.98(d, J=8 Hz, 1H), 7.93-7.89 (m, 2H), 7.28-7.23 (m, 2H), 7.22 (s, 1H),7.12 (d, J=8 Hz, 1H), 5.07 (quin, J=8 Hz, 1H), 4.14 (t, J=8 Hz, 2H),3.85 (m, 2H), 3.36 (m, 2H), 3.09 (t, J=8 Hz, 2H), 2.80 (m, 1H),1.57-1.66 (m, 4H), 1.41 (d, J=7 Hz, 3H).

Synthetic Method 3:

Add TEA (65 mL, 468 mmol) to a mixture of[5-(1-aminoethyl)-2,3-dihydro-1H-indol-1-yl](tetrahydro-2H-pyran-4-yl)methanonehydrochloride, Isomer 1 (Preparation 7B) (70 g, 225 mmol) in DCM (700mL) at 0-5° C. Add 4-fluorobenzoyl chloride (37.85 g, 239 mmol)dropwise. Warm the mixture to room temperature and stir for two hours.

Add water dropwise at a rate to keep the temperature below 30° C. andstir the mixture at 20-30° C. for one hour. Separate the layers and washthe organic layer with 18% aqueous H₂SO₄. Separate the layers and washthe organic layer with 7% aqueous NaHCO₃. Separate the layers and washthe organic layer with water. Separate the layers and then pass theorganic solution through a carbon filter. Treat the solution withSI-Thiol (7 g) and heat the mixture to 40° C. Stir the resulting mixturefor 12 hours. Cool the mixture to room temperature and filter themixture through diatomateous earth. Concentrate the organic layer to 200mL (˜3 vols). Add acetone (140 mL, 2 vols) and concentrate the resultingmixture to 200 mL (˜3 vols). Treat with additional acetone (280 mL, 4vols) and water (280 mL, 4 vols). Heat at 65° C. for two hours untilreaction is a clear solution. Cool slowly to 30° C. over three hours.Stir at 30° C. for one hour. Add water (140 mL, 2 vols) dropwise andcontinue stirring at 30° C. for one hour. Cool slowly to 3-8° C. overapproximately two hours. Stir at this temperature for six hours. Filterand rinse the solids with water (140 mL, 2 vols). Dry the solids at 55°C. for four to six hours. Obtain the desired product as a white solid(55 g, 61.6%).

X-Ray Powder Diffraction Collection Procedure for Example 1A

The XRD patterns of crystalline solids are obtained on a Bruker D4Endeavor X-ray powder diffractometer, equipped with a CuKa source(λ=1.54060 Δ) and a Vantec detector, operating at 35 kV and 50 mA. Thesample is scanned between 4 and 40° in 2θ, with a step size of 0.0087°in 2θ and a scan rate of 0.5 seconds/step, and with 0.6 mm divergence,5.28 mm fixed anti-scatter, and 9.5 mm detector slits. The dry powder ispacked on a quartz sample holder and a smooth surface is obtained usinga glass slide. Collect the crystal form diffraction patterns at ambienttemperature and relative humidity.

X-Ray Powder Diffraction Collection Procedure for 1A Method 3

A prepared sample of Example 1A (Synthetic method 3) is characterized byan XRD pattern using CuKa radiation as having diffraction peaks (2-thetavalues) as described in Table 1 below. Specifically the pattern containsa peak at 17.38° in combination with one or more of the peaks selectedfrom the group consisting of 12.51°, 15.65°, 16.37°, 17.56°, 21.48° and25.23° with a tolerance for the diffraction angles of 0.2 degrees(2θ±0.2°).

TABLE 1 X-ray powder diffraction peaks of Example 1A method 3 Peak Angle(2-Theta °) Intensity (%) 1 9.99 13 2 12.51 73 3 15.65 90 4 16.37 57 517.38 100 6 17.56 62 7 18.79 25 8 19.81 38 9 21.48 56 10 23.38 43 1124.41 21 12 24.70 17 13 25.23 64 14 25.46 28 15 27.69 15

Determination of Absolute Configuration for Example 1A

Prepare a single crystal of4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamideby suspending 10 mg of4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamidein 1:1 EtOH/heptane (1.75 mL) and slurrying on an orbital shaker forthree days. Use a colorless bladed-like specimen of4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,approximate dimensions 0.020 mm×0.080 mm×0.300 mm, for the X-raycrystallographic analysis. Measure the X-ray intensity data using an IμCuKα radiation source (λ=1.54178 Δ) and a Bruker D8 Venture based3-circle goniometer diffractometer equipped with Photon 100 SL areadetector. Collect a total of 8840 frames. Integrate the frames with theBruker SAINT software package using a narrow-frame algorithm. Theintegration of the data using a monoclinic unit cell yielded a total of7242 reflections to a maximum 0 angle of 68.28° (0.83 Δ resolution), ofwhich 3059 are independent (average redundancy 2.367,completeness=95.9%, Rint=5.83%, Rsig=6.58%) and 2893 (94.57%) is greaterthan 2σ(F²). The final cell constants of a=5.5831(13) Å, b=5.1082(9) Å,c=35.013(6) Å, β=90.578(17)°, volume=998.5(3) Å³, are based upon therefinement of the XYZ-centroids of 6280 reflections above 20 σ(I) with10.09°<2θ<136.8°. Correct the data for absorption effects using themulti-scan method (SADABS). The ratio of minimum to maximum apparenttransmission is 0.784. The calculated minimum and maximum transmissioncoefficients (based on crystal size) are 0.8020 and 0.9850. Solve thestructure and refine using the Bruker SHELXTL Software Package, usingthe space group P 2₁, with Z=2 for the formula unit, C₂₃H₂FN₂O₃. Thefinal anisotropic full-matrix least-squares refinement on F2 with 264variables converge at R1=9.17%, for the observed data and wR2=23.48% forall data. The goodness-of-fit is 1.141. The largest peak in the finaldifference electron density synthesis is 0.506 e−/Δ³ and the largesthole is −0.358 e−/Δ³ with an RMS deviation of 0.088 e−/Δ³. On the basisof the final model, the calculated density is 1.319 g/cm³ and F(000),420 e−. The absolute structure parameter refines to 0.12(16), verifyingthe stereochemistry of the chiral center. The absolute structure isdetermined to be the R-configuration at the stereocenter.

Example 1B4-Fluoro-N-{(1S)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,Isomer 2

Purify racemic4-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide(Example 1) by chiral chromatography to afford the second elutingenantiomer as the title compound. ES/MS (m/z): 397.0 (M+H). Purificationconditions: Column: CHIRALPAK® AD-H, 21×150 mm; Mobile phase: 40% MeOHin CO₂; Column temperature: 40° C.; Flow rate: 70 g/minute; UVW: 225 nm.Confirm enantiomeric enrichment of Isomer 2 by chiral analytical SFC(98.3% ee, R_(t): 2.37 minutes; Column: CHIRALPAK® AD-H, 4.6×150 mm;Mobile phase: 40% MeOH in CO₂; Flow rate: 5 mL/minute; UVW: 225 nm). ¹HNMR (d₆-DMSO) δ 8.73 (d, J=8 Hz, 1H), 7.98 (d, J=8 Hz, 1H), 7.93-7.89(m, 2H), 7.28-7.23 (m, 2H), 7.22 (s, 1H), 7.12 (d, J=8 Hz, 1H), 5.07(quin, J=8 Hz, 1H), 4.14 (t, J=8 Hz, 2H), 3.85 (m, 2H), 3.36 (m, 2H),3.09 (t, J=8 Hz, 2H), 2.80 (m, 1H), 1.57-1.66 (m, 4H), 1.41 (d, J=7 Hz,3H).

Prepare Example 2 Essentially Analogous to Example 1, Using the StartingMaterial from Preparation 3

Ex No. Chemical Name Structure Physical data 2 Racemic 4-chloro-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indo1-5-yl]ethyl}benzamide

ES/MS (m/z): 413.0 (M + H)

Example 2A and 2B4-Chloro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,Isomer 1 and4-Chloro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,Isomer 2

Purify racemic4-chloro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide(Example 2) by chiral SFC to afford the first eluting enantiomer (Isomer1). ES/MS (m/z): 413.0 (M+H). Purification conditions: CHIRALPAK® AD-H,21×150 mm; Mobile phase: 40% iPrOH in CO₂; Flow rate: 70 g/minute; UVW:260 nm. Confirm enantiomeric enrichment of Isomer 1 by chiral analyticalSFC (>99% ee, R_(t)=1.97 minutes; Column: CHIRALPAK® AD-H, 4.6×150 mm;Mobile phase: 40% iPrOH in CO₂; Column temperature: 40° C.; Flow rate: 5mL/minute; UVW: 225 nm).

The above purification also yields the second eluting enantiomer (Isomer2). ES/MS (m/z): 413.0 (M+H). Confirm enantiomeric enrichment of Isomer2 by chiral analytical SFC (>99% ee, R_(t): 3.04 minutes; Column:CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase: 40% iPrOH in CO₂; Flow rate:5 mL/minute; UVW: 225 nm).

Prepare Examples 3 Through Example 9 Essentially Analogous to Example 1,Using the Starting Material from Preparation 3

Ex No. Chemical Name Structure Physical data 3 Racemic 4-cyano-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

ES/MS (m/z): 404.4 (M + H) 4 Racemic N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3- dihydro-1H-indol-5- yl]ethyl}benzamide

ES/MS (m/z): 379.4 (M + H) 5 Racemic 4-methyl-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

ES/MS (m/z): 393.4 (M + H) 6 Racemic 4-chloro-3-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

ES/MS (m/z): 431.4 (M + H) 7 Racemic 3-chloro-4-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

ES/MS (m/z): 431.4 (M + H) 8 Racemic 4-ethenyl-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

ES/MS (m/z): 405.4 (M + H) 9 Racemic 2,4-difluoro-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide

ES/MS (m/z): 415.0 (M + H)

Example 9A and B2,4-Difluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,Isomer 1 and2,4-Difluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,Isomer 2

Purify racemic2,4-difluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide(Example 9) by chiral SFC to afford the first eluting enantiomer (Isomer1). ES/MS (m/z): 415.0 (M+H). Purification conditions: CHIRALPAK® AD-H,21×150 mm; Mobile phase: 40% MeOH in CO₂; Column temperature: 40° C.;Flow rate: 70 g/minute; UVW: 225 nm. Confirm enantiomeric enrichment ofIsomer 1 by chiral analytical (98.6% ee, R_(t): 1.72 minutes; Column:CHIRALPAK® AD-H, 4.6×150 mm; Mobile phase: 40% MeOH in CO₂; Flow rate: 5mL/minute; UVW: 225 nm).

The above purification also yields the second eluting (Isomer 2). ES/MS(m/z): 415.2 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical SFC (98.5% ee, R_(t): 2.60 minutes; Column: CHIRALPAK®AD-H, 4.6×150 mm; Mobile phase: 40% MeOH in CO₂; Flow rate: 5 mL/minute;UVW: 225 nm).

Prepare Examples 10 Through Example 13 Essentially Analogous to Example1, Using the Starting Material from Preparation 7

Ex No. Chemical Name Structure Physical data 104-(Difluoromethyl)-N-{1-[1- (tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H- indol-5-yl]ethyl}benzamide, Isomer A

ES/MS (m/z): 429.0 (M + H) 11 4-(Fluoromethyl)-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide, Isomer A

ES/MS (m/z): 411.2 (M + H) 12 2-(Benzyloxy)-4-(fluoro)-N-{1-[1-(tetrahydro-2H-pyran- 4-ylcarbonyl)-2,3-dihydro- 1H-indol-5-yl]ethyl}benzamide, Isomer A

ES/MS (m/z): 503.2 (M + H) 13 4-Fluoro-2-[(2- hydroxyethyl)amino]-N-{1-[1-(tetrahydro-2H-pyran-4- ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide, Isomer A

ES/MS (m/z): 456.2 (M + H)

Prepare Examples 14 and 15 Essentially Analogous to Example 1A,Synthetic Method 2

Ex No. Chemical Name Structure Physical data 144-Fluoro-N-{2-[1-(tetrahydro- 2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5- yl]propan-2-yl}benzamide

ES/MS (m/z): 411.2 (M + H) 15 4-Cyano-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3- dihydro-1H-indol-5- yl]propyl}benzamide,Isomer A

ES/MS (m/z): 418.2 (M + H)

Example 16 Diastereomeric4-Fluoro-N-[1-{1-[tetrahydro-2H-pyran-3-ylcarbonyl]-2,3-dihydro-1H-indol-5-yl}ethyl]benzamide(Mix of 2 Diastereomers)

Treat a mixture ofN-[1-(2,3-dihydro-H-indol-5-yl)ethyl]-4-fluorobenzamide, Isomer 1(Preparation 24A) (150 mg, 0.528 mmol), racemictetrahydropyran-3-carboxylic acid (100 mg. 0.739 mmol) andN,N-diisopropylethylamine (0.277 mL, 1.58 mmol) in DCM (5.28 mL) with1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexafluorophosphate (304 mg, 0.791 mmol). Stir at room temperature for45 minutes. Dilute the reaction mixture with DCM. Add water andsaturated aqueous sodium bicarbonate solution. Separate the layers andextract the aqueous layer twice with DCM. Pass the combined organiclayer through a hydrophobic frit (ISOLUTE® phase separator cartridge)and concentrate the filtrate. Purify by silica gel column chromatographyeluting with a gradient of 20-55% acetone in hexanes to give the titlecompound (184 mg, 88%) as a white solid. ES/MS (m/z): 397.2 (M+H).

Example 16A and B4-Fluoro-N-[1-{1-[tetrahydro-2H-pyran-3-ylcarbonyl]-2,3-dihydro-1H-indol-5-yl}ethyl]benzamide,Isomer 1 and4-Fluoro-N-[1-{1-[tetrahydro-2H-pyran-3-ylcarbonyl]-2,3-dihydro-1H-indol-5-yl}ethyl]benzamide,Isomer 2

Purify diasteromeric4-fluoro-N-[1-{1-[tetrahydro-2H-pyran-3-ylcarbonyl]-2,3-dihydro-1H-indol-5-yl}ethyl]benzamide(Example 16) by chiral chromatography to afford the first elutingdiastereomer (Isomer 1). MS (m/z): 397.2 (M+H). Purification conditions:CHIRALCEL® OJ-H, 30×250 mm; Mobile phase: 100% MeOH; Flow rate: 30mL/minute; UVW: 225 nm. Confirm enantiomeric enrichment of Isomer 1 bychiral analytical HPLC (>99% de, R_(t): 3.42 minutes; Column: CHIRALCEL®OJ-H, 4.6×150 mm; Mobile phase: 100% MeOH (containing 0.2%isopropylamine); Flow rate: 1 mL/minute; UVW: 225 nm).

The above purification also yields the second eluting (Isomer 2). ES/MS(m/z): 397.2 (M+H). Confirm enantiomeric enrichment of Isomer 2 bychiral analytical HPLC (97.8% de, R_(t): 4.66 minutes; Column CHIRALCEL®OJ-H, 4.6×150 mm; Mobile phase: 100% MeOH (containing 0.2%isopropylamine); Flow rate: 1 mL/minute; UVW: 225 nm).

Example 174-Fluoro-2-hydroxy-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,Isomer A

Add 10% Pd/C (10 mg) to a nitrogen flushed solution of2-(benzyloxy)-4-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamide,Isomer A (Example 12) (96.0 mg, 0.19 mmol) in ethanol (2 mL) andhydrogenate with 1 atm (101 kPa) of hydrogen at room temperature for onehour. Filter over diatomaceous earth and concentrate the filtrate toobtain the desired compound (66 mg, 84%) as a white solid. ES/MS (m/z):413.0 (M+H).

Example 18 Racemic4-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propyl}benzamide

Treat a mixture of racemicN-[1-(2,3-dihydro-1H-indol-5-yl)propyl]-4-fluorobenzamide (Preparation22) (200 mg, 0.650 mmol) in DCM (6.5 mL) with N,N-diisopropylethylamine(0.228 mL, 1.30 mmol). Add tetrahydropyran-4-carbonyl chloride (110 mg,0.715 mmol) and stir for 30 minutes. Dilute the reaction mixture withDCM. Add water and saturated aqueous sodium bicarbonate solution.Separate the layers and extract the aqueous layer twice with DCM. Passthe combined organic layer through a hydrophobic frit (ISOLUTE® phaseseparator cartridge) and concentrate the filtrate. Purify by silica gelcolumn chromatography eluting with a gradient of 20-60% acetone inhexanes to give the title compound as a light peach-colored foam (244mg, 91%). ES/MS (m/z): 411.2 (M+H).

Example 18A and B4-Fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propyl}benzamide,Isomer 1 and4-Fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propyl}benzamide,Isomer 2

Purify racemic4-fluoro-N-{1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]propyl}benzamide(Example 18) by chiral SFC chromatography to afford the first elutingenantiomer (Isomer 1). ES/MS (m/z): 411.2 (M+H). Purificationconditions: CHIRALPAK® AS-H, 21×150 mm column; Mobile phase: 25% MeOH inCO₂; Column temperature: 40° C.; Flow rate: 80 g/minute; UVW: 260 nm.Confirm enantiomeric enrichment of Isomer 1 by chiral analytical SFC(>99% ee, R_(t): 0.92 minutes; Column: CHIRALPAK® AS-H, 4.6×150 mm;Mobile phase: 25% MeOH in CO₂; Flow rate: 5 mL/minute; UVW: 225 nm).

The above purification also yields the second eluting enantiomer (Isomer2). ES/MS (m/z): 411.2 (M+H). Confirm enantiomeric enrichment of Isomer2 by chiral analytical SFC (>99% ee, R_(t): 1.53 minutes; Column:CHIRALPAK® AS-H, 4.6×150 mm; Mobile phase: 25% MeOH in CO₂; Flow rate: 5mL/minute; UVW: 225 nm).

Reference Example 11-(2,4-Difluorophenyl)-3-{[1-(3,4,5-tritritiobenzoyl)-2,3-dihydro-1H-indol-5-yl]methyl}urea

In a tritaration flask, stir1-(2,4-difluorophenyl)-3-{[1-(3,4,5-tribromobenzoyl)-2,3-dihydro-1H-indol-5-yl]methyl}urea(3 mg, 0.005 mmol), palladium (10% on carbon, 3 mg) andN,N-diisopropylethylamine (10 μL, 0.06 mmol) in DMF (1 mL) under 3 Ci oftritium for three hours. Filter the reaction and co-evaporate thefiltrate with EtOH to remove the labile tritium. Dissolve the residue inEtOH and purify by reverse phase column chromatography (Column: GEMINI®C18 250×10 mm; Mobile phase: A: water/TFA (1000:1), B: ACN/TFA (1000:1);gradient: 20-70% B over 60 minutes; flowrate 3 mL/minute) to give thetitle compound which was dissolved in EtOH. MS: 414.19 (M+H) and 74Ci/mmol.

Reference Example 21-(2,4-Difluorophenyl)-3-{[1-(phenylcarbonyl)-2,3-dihydro-1H-indol-5-yl]methyl}urea

Dissolve 1-(2,4-difluorophenyl)-3-(2,3-dihydro-1H-indol-5-ylmethyl)urea(300 mg, 0.99 mmol) in DCM (20 mL) and add benzoyl chloride (0.13 mL,1.1 mmol) and TEA (0.27 mL, 1.9 mmol). Stir the reaction mixture at roomtemperature for two hours. Concentrate and purify the residue by reversephase purification (Column: Redisep Rf Gold High Performance C18 ReversePhase Column; Mobile phase: A: 0.1% formic acid in water, B: ACN;gradient: 0-80% B over 30 minutes; flow rate: 60 mL/minute, UVW: 219/254nm) and isolate the product by lyophilization to give the title compound(403 mg, 79%). ES/MS (m/z): 408.2 (M+H).

The immune system is a critical checkpoint that restrains tumordevelopment. As such, cancers have evolved many mechanisms to evade,suppress, or otherwise subvert the immune system. While tryptophan isabsolutely essential for cancer cell growth, its degradation is selectedfor in a broad array of cancers through the expression of indoleamine2,3 dioxygenase (IDO1) either by the cancer cell itself (intrinsic), orby cells in the microenvironment or tumor draining lymph nodes (TDLNs)(extrinsic). The selective activation of IDO1 in the tumormicroenvironment while counter to rapid cell growth provides the tumorwith an effective strategy to avoid immunosurveillance, a criticalcheckpoint in cancer development and resistance to therapy. Theimmunosuppressive activity of IDO1 is a direct result of the localdepletion of tryptophan and the concomitant production of kynurenine,both of which are immunosuppressive.

The immunosuppressive role of IDO1 activity impacts multiple cell typesincluding cell suppression [T-cells (Frumento, et al. (2002) J Exp Med196(4): 459-468; Terness, et al. (2002) J Exp Med 196(4): 447-457) andNK cells (Della Chiesa, et al. (2006) Blood 108(13): 4118-4125)], celldevelopment [regulatory T-cells (Sharma, et al. (2007) J Clin Invest117(9): 2570-2582; Chen, et al. (2008) J Immunol 181(8): 5396-5404;Baban, et al. (2009) J Immunol 183(4): 2475-2483)] and suppressiveantigen presenting cells [suppressive dendritic cells and macrophages(Munn, et al. (2004) J Clin Invest 114(2): 280-290; Munn, et al. (2005)Immunity 22(5): 633-642; Sharma, et al. (2007) J Clin Invest 117(9):2570-2582)], and recruitment and expansion [myeloid-derived suppressorcells (Yu, et al. (2014) J Immunol 193(5): 2574-2586; Holmgaard, et al.(2015) Cell Rep 13(2): 412-424)]. IDO1 activity exhibits these effectsthrough depletion of tryptophan and the concomitant increase inkynurenine in the tumor, the tumor microenvironment and TDLNs.

Both the local depletion of tryptophan and the production of kynurenineby IDO1 expression in the tumor microenvironment or in TDLNs support thedevelopment and activation of Tregs (Sharma, et al. (2007) J Clin Invest117(9): 2570-2582), MDSCs (Holmgaard, et al. (2015) Cell Rep 13(2):412-424), and regulatory dendritic cells (Sharma, et al. (2007) J ClinInvest 117(9): 2570-2582) all of which play immunosuppressive roles thatsupport tumor growth. The depletion of tryptophan supports Tregdevelopment through the activation of the stress response kinase GCN2,which is stimulated in response to the accumulation of uncharged tRNAs.T-cells lacking GCN2 are not susceptible to IDO1-mediated inhibition ofproliferation or the induction of an anergic phenotype (Munn, et al.(2005) Immunity 22(5): 633-642). In addition to tryptophan depletion,IDO1 activity leads to high concentrations of the downstream metabolitekynurenine, an important immunosuppressive molecule. Similar totryptophan depletion, the activation of aryl hydrocarbon receptor (AHR)by kynurenine is essential for the generation of regulatory T-cells(Mezrich, et al. (2010) J Immunol 185(6): 3190-3198), and elevatedproduction of kynurenine and expression of AHR correlate with poorprognosis in human brain cancer (Opitz, et al. (2011) Nature 478(7368):197-203). Kynurenine blocks T-cell and NK cell proliferation (Boyland,et al. (1956) Biochem J 64(3): 578-582) and is an agonist of the AHRreceptor (Mezrich, et al. (2010) J Immunol 185(6): 3190-3198; Opitz, etal. (2011) Nature 478(7368): 197-203), a transcription factor thatregulates innate immune-mediated production of cytokines such as IL-1,IL-6 and IL-21, and is overexpressed in several cancers where it isthought to facilitate tumor progression and resistance to therapy(Julliard, et al. (2014) Front Immunol 5: 458). In fact, the intrinsicexpression of IDO1 in cancer is regulated, in part, bykynurenine-mediated activation of an AHR-IL-6-STAT3 signaling loop thatenforces the expression of IDO1 and inhibits T-cell proliferation.Expression of this IDO1 signaling axis is associated with a reduction inrelapse free survival in lung cancer patients (Litzenburger, et al.(2014) Oncotarget 5(4): 1038-1051). IDO1-mediated IL-6 production alsoplays an important role in supporting the development of pro-tumorigenicMDSCs and disruption of IDO1 reduced IL-6 production, attenuated MDSCsuppressive activity, delayed tumor growth and increased survival in aKRAS-induced lung cancer model (Smith, et al. (2012) Cancer Discov 2(8):722-735). The connection between IDO1-dependent depletion of tryptophanand kynurenine-dependent activation of AHR provides a lynch pinexplaining why tryptophan catabolism is intimately associated withimmune escape, a critical checkpoint that restrains cancer progression.

The regulation of IDO1 expression in the tumor microenvironment iscomplex. IDO1 was the first IFN-γ-regulated gene discovered (Yoshida, etal. (1981) Proc Natl Acad Sci USA 78(1): 129-132). In fact, there is astrong correlation between IFN-γ and IDO1 expression across all cancerhistologies (http://cancergenome.nih.gov/). Additionally, IDO1expression is upregulated by type I interferons, TLR ligands, TNF, IL-1,CTLA-4, CD200, GITR, CD40 and TGF-β, all of which play important rolesin the immune system, and cancer development, progression and responseto therapy. High IDO1 activity as measured by IDO1 expression,tryptophan depletion and/or increase in kynurenine has been implicatedin the poor prognoses, reduced survival rates and increased metastaticpotential of a wide variety of tumor types. As such, increases in serumlevels of kynurenine with a concomitant reduction in tryptophan areevidenced in breast, colorectal cancer, head and neck, lung and prostatecancers (Liu, et al. (2010) Blood 115(17): 3520-3530). In addition, IDO1is chronically activated in cancer patients (Schrocksnadel, et al.(2006) Clin Chim Acta 364(1-2): 82-90), associated with extensivedisease (Huang, et al. (2002) Br J Cancer 86(11): 1691-1696), pooroutcome and/or resistance to standard chemotherapy in several cancersincluding melanoma (Weinlich, et al. (2007) Dermatology 214(1): 8-14),acute myeloid leukemia (Chamuleau, et al. (2008) Haematologica 93(12):1894-1898; Corm, et al. (2009) Leuk Res 33(3): 490-494), breast andcervical cancer (Inaba, et al. (2010) Gynecol Oncol 117(3): 423-428; Yu,et al. (2011) Clin Dev Immunol 2011: 469135; Yu, et al. (2013) J Immunol190(7): 3783-3797; Chen, et al. (2014) Breast Cancer Res 16(4): 410):Clin Cancer Res. 2007 Dec. 1; 13(23):6993-7002; Trott, et al. (2016).Oncotarget, 7(41), 66540-66557, colorectal cancer, renal cell carcinoma,cutaneous melanoma, diffuse large B-cell lymphoma, endometrial cancer,gastric cancer, glioma, hepatocellular carcinoma, Hodgkin's lymphoma,laryngeal squamous cell carcinoma, lung cancer, multiple myeloma,Non-Hodgkin's lymphoma, esophageal and oral squamous cell carcinoma,osteosarcoma, ovarian cancer, pancreas ductal carcinoma, T-cell leukemiaand thyroid carcinoma. IFN-γ is a critical effector cytokine secretedfrom activated NK and T-cells. Negative regulatory circuits that areengaged to restrain T-cell activity either systemically (CTLA-4) orlocally (PD-L1/L2) are currently approved for use as anti-cancer agentswhere they enhance T-cell-mediated tumor growth inhibition. Geneticknockouts of checkpoints such as CTLA-4, PD-1 or PD-L result in themarked enhancement of IFN-γ production (Latchman, et al. (2004) ProcNatl Acad Sci USA 101(29): 10691-10696; Pandiyan, et al. (2007) JImmunol 178(4): 2132-2140), which can engage the immunosuppressiveIFN-γ-to-IDO1 axis. The inhibition of intrinsic IDO1 expression with1-Methyl Tryptophan in a mouse melanoma model, significantly improvedthe efficacy of Ipilimumab, a CTLA-4 blocking antibody (Holmgaard, etal. (2013) J Exp Med 210(7): 1389-1402). This enhanced efficacy ofIpilimumab was associated with an increase in CD8 effector cells and adecrease in Tregs. These observations were extended to other antibodiestargeting PD1, PD-L1 and GITR where the inhibition of IDO1 enhancedtheir anti-cancer activity. Mechanistically IDO1 was shown to impede theefficacy of these checkpoint inhibitors through the induction of Tregswith the subsequent recruitment of MDSCs creating an immunosuppressiveenvironment at the tumor (Holmgaard, et al. (2015) Cell Rep 13(2):412-424). Immunotherapeutic approaches to treat cancer such as IFN-γitself, innate immune activators such as CpG-ODNs, anti-4-1BB (CD137),anti-OX40, anti-PD-1/PD-L1/PD-L2, anti-CTLA 4 all have the potential toactivate IDO1 expression restraining their long-term efficacy in theclinic. Therefore, there may be significant therapeutic potential incombining IDO1 inhibitors with these agents. Specifically, combinationof IDO1 inhibitors with anti-PD1 antibodies (pidilizumab, nivolumab,pembrolizumab), anti-PD-L1 antibodies (durvalumab, atezolizumab,avelumab), anti-CTLA-4 antibodies (ipilimumab), anti-OX40 antibodies(MEDI6469, KHK4083) and anti-4-1BB (CD137) antibodies (PF-05082566) havesignificant therapeutic potential in a wide range of tumor types.

Taken together, these data suggest that inhibitors of tryptophandepletion and the concomitant production of kynurenine such as IDO1inhibitors may be useful as a single agent or in combination in avariety of cancer types in both treatment naïve as well as treatmentresistant cancer patients. This utility has been demonstrated by knownIDO1 inhibitors such as epacadostat (INCB024360) and NLG919. Epacadostatis known to bind to IDO1 and block the catabolism of tryptophan and thesubsequent production of kynurenine both in vitro and in vivo.Additionally, epacadostat has demonstrated single agent efficacy inpre-clinical mouse models, CT26 and PAN02, an effect that is dependentupon the presence of T-cells. (Yue, et al. (2009) J Med Chem 52(23):7364-7367; Koblish, et al. (2010) Mol Cancer Ther 9(2): 489-498; Liu, etal. (2010) Blood 115(17): 3520-3530; Jochems, et al. (2016) Oncotarget,Advance Publications). The pre-clinical efficacy of epacadostat hastranslated into human clinical trial outcomes (NCT11195311).

The results of the following assays demonstrate evidence that thecompounds exemplified herein are useful as kynurenine productioninhibitors such as IDO1 inhibitors and may be useful in treating cancer.Furthermore, the results of the following assays demonstrate thatcertain exemplified compounds bind to IDO1 and that all exemplifiedcompounds inhibit the conversion of tryptophan to kynurenine in cancercells both in vitro and in vivo.

Binding Assay for IDO1

The purpose of this assay is to demonstrate that certain exemplifiedcompounds bind to IDO1 in vitro. Specifically, this assay assesses theability of test compounds to compete with a tritiated spy molecule1-(2,4-difluorophenyl)-3-[[1-(3,4,5-tritritiobenzoyl)indolin-5-yl]methyl]ureaand allows for the calculation of the binding affinity, IC₅₀.

Competitive Binding of1-(2,4-Difluorophenyl)-3-[[1-(3,4,5-tritritiobenzoyl)indolin-5-yl]methyl]ureato IDO1

Load 300 nM His₆-IDO1 (Proteros, SwissProtID P14902, Cat # PR-0278,batch 19/59, 98 mg/mL in 25 mM MES pH 6.5, 150 mM KCl, purity >95%)diluted in DPBS to each well of nickel coated plate (Sigma, Cat # S5563)and incubate overnight at 4° C. Remove unbound proteins by washing platewith 300 μL DPBS four times in BIOTEK® Microplate Washer. Add 100 μL ofblocking buffer (0.05% TWEEN® 20/DPBS) per well and incubate for onehour at room temperature to reduce nonspecific binding. While blockingthe plate, prepare 50 nM1-(2,4-difluorophenyl)-3-[[1-(3,4,5-tritritiobenzoyl)indolin-5-yl]methyl]urea(Biocair, Cat # TRQ41455) by diluting in DPBS, and serially diluteunlabeled stock solution 2.5-fold in DMSO to generate an eleven pointdilution curve. Add 5 μL of serial diluted unlabeled compounds to 95 μLof 50 nM1-(2,4-difluorophenyl)-3-[[1-(3,4,5-tritritiobenzoyl)indolin-5-yl]methyl]urea,add mixture to the wells in the plate, and incubated at room temperaturefor four hours with gentle shaking. To determine the maximumdisplacement of the tritiated-spy molecule(1-(2,4-difluorophenyl)-3-[[1-(3,4,5-tritritiobenzoyl)indolin-5-yl]methyl]urea),add an excess amount of unlabeled1-(2,4-difluorophenyl)-3-{[1-(phenylcarbonyl)-2,3-dihydro-1H-indol-5-yl]methyl}urea(ChemDiv, Cat # G714-0242)) 100 μM to 50 nM1-(2,4-difluorophenyl)-3-[[1-(3,4,5-tritritiobenzoyl)indolin-5-yl]methyl]ureaand add to nonspecific binding control wells in the plate. After fourhour incubation, aspirate wells using a MultiMek96 and wash the platequickly once with 300 μL ice-cold DPBS using a BIOTEK® Microplatewasher. Add 100 μL of 100 mM imidazole in PBS pH 7.5 to each well andincubated for 10 minutes at room temperature to displace IDO1-ligandcomplex from the nickel-coated plate. Transfer displaced IDO1-ligandcomplex into a 96-well white clear bottom plate (Costar, Cat #3632)containing 200 μL of Microscint-20 (Perkin Elmer, Cat #6013621), perwell using a MultiMek96. Quantitate total bound and nonspecific binding(NSB) of the1-(2,4-difluorophenyl)-3-[[1-(3,4,5-tritritiobenzoyl)indolin-5-yl]methyl]urealigand using a Trilux Microbeta Counter. Use total bound and NSB valuesto calculate the IC₅₀ for unlabeled compound using nonlinear regressionanalysis in GraphPad Prism. The results of this assay demonstrate thatcertain exemplified compounds bind to IDO1. For example, Examples 1A and1B demonstrate IC₅₀ values less than 1.5 μM. Specifically, the IC₅₀ forExample 1A is 0.033 μM±0.0028 (n=2).

Kynurenine Production Assay (SKOV3)

The purpose of this assay is to evaluate the inhibition of theproduction of kynurenine, N-formyl-kynurenine and the depletion oftryptophan in IDO1 expressing cancer cells and assess whether compoundsare overtly toxic to these cells. Exemplary compounds are tested for theinhibition of IDO1 activity in SKOV3 (ATCC, Cat # HTB-77), an ovariancancer cell line that intrinsically expresses IDO1. Due to IDO1expression, SKOV3 cells degrade tryptophan with the concomitantproduction of kynurenine and compounds are tested for their ability toinhibit the production of kynurenine, N-formyl-kynurenine and thedepletion of tryptophan. Optionally, overt toxicity of compounds can beassessed by monitoring cell viability.

Synthesis of Internal Standards Synthesis of N-Formyl L-Kynurenine-d4(2S)-2-amino-4-oxo-4-(2,3,4,5-tetradeuterio-6-formamido-phenyl)butanoicacid

Add a preformed mixture of acetic anhydride (0.026 mL, 0.264 mmol) informic acid (0.052 mL, 1.32 mmol) to a mixture of L-kynurenine-d4 (56mg, 0.264 mmol) in formic acid (0.132 mL). Stir the resulting mixture atroom temperature under nitrogen for two hours. Dilute the reactionmixture with ACN and concentrate under a stream of nitrogen. Purify theresidue by reversed-phase HPLC (Column: PHENOMENEX® LUNA® 5 μm C18 (2)100Δ AXIA, 30×75 mm; eluent: A: 0.1% formic acid/water, B: 0.1% formicacid/ACN; gradient: 0% B for 2 minutes then gradient to 22% B over 5minutes; flow: 85 mL/minutes at UVW 231/214 nm) to give the titlecompound 29 mg as a fluffy white solid after lyophilization. ES/MS(m/z): 241.0 (M+H).

Cell Treatment and Cell Viability

Plate SKOV3 cells, an IDO1-expressing ovarian cancer cell line, at20,000 cells per well in 100 μL of McCoys 5A media (Gibco, Cat#16600-082) supplemented with non-essential amino acids (Gibco, Cat#11140-050), 1 mM sodium pyruvate (Gibco, Cat #11360-070), and 10% fetalbovine serum, complete media, in a 96 well tissue culture plate (BDBiosciences). Then, incubate cells for 16 hours in a 37° C. incubatorwith 5% CO₂. Prepare compound serial dilutions from 10 mM stock testcompounds in DMSO. Serially dilute the stock solution 3-fold in DMSO,and transfer 5 μL of compounds to an intermediate dosing platecontaining 95 μL of complete media to generate a ten-point dilutioncurve with final compound concentrations ranging from either 1 μM to 0.5μM or 10 μM to 0.5 nM. Decant the media from the plate containing cellsand blot onto paper towels. Wash plate twice with 90 μL of completemedia per well and replace the final wash with 90 μL of complete media.After washing, add 10 μL of serial diluted compounds from theintermediate dosing plate to each well of the plate(s) and incubate for18 hours in a 37° C. incubator with 5% CO₂. The final DMSO concentrationin the assay is 0.5%. At the end of the 18 hours incubation, transfer 50μL of media from each well into a 96 well v-bottom plate (ThermoScientific), seal the plate, and store at −80° C. for subsequent massspectrometric-based measurement of kynurenine, N-formyl-kynurenine andtryptophan. Optionally, return original plate(s) to the incubator for anadditional 24 hours and measure the viability of cells by adding anequal volume of CELLTITER-GLO® (Promega) and measure luminescence in anPERKIN ELMER® EnVision plate reader.

Mass Spectrometric (MS) Measurement of Tryptophan, N-Formyl-Kynurenine,and Kynurenine

Thaw samples collected from SKOV3 cell-based assay on ice and clear anycellular debris by centrifuging plate at 3220×g for one minute at 4° C.Add 12.5 μL of internal standards consisting of 2.5 μg/mLL-tryptophan-2′,4′,5′,6′,7′-d5 (CDN Isotopes, Cat # D-1522),L-kynurenine sulfate-ring-d4,3,3-d2 (Cambridge Isotope Laboratories, Cat# DLM-7842-0.01) and internally prepared N-formyl L-kynurenine-d4. Heatseal all plates with Easy Peel seals (ThermoScientific) and mix byvortexing for 1-2 minutes and then centrifuge for one minute at 3220×gat 4° C. Generate standard calibration solutions for quantification ofkynurenine and N-formylkynurenine by dissolving each in water to give afinal concentration of 1 mg/mL. Aliquot 20.8 μL kynurenine and 23.6 μLN-formylkynurenine from their respective 1 mg/mL stock and dilute to 1mL using McCoys 5A media to give a final concentration for each standardof 100 μM. Serial dilute calibration solution 2-fold in complete mediato obtain a 5-point standard curve with final concentrations of 5 μM to0.313 μM (kynurenine) and 2 μM to 0.125 μM (N-formylkynurenine). Inject1 μL of media sample (unknown) or standard calibration solution onto anLC/MS-MS system consisting of a SHIMADZU® Prominence 30A HPLC system andan AB SCIEX® 5500 triple quadrupole mass spectrometer. Separate analyteson a XBridge™ C18 column, 2.1×50 mm, 3.5 μm (Waters, Cat #186003021)maintained at 35° C., with mobile phase flow rate of 0.7 mL/minute. Themobile phase A is 0.1% formic acid in water, and mobile phase B is MeOH.The gradient profile is: 0 minutes, 0.5% B; 0.8 minutes, 98% B; 1.10minutes, 98% B; 1.11 minutes, 0.5% B; 1.7 minutes, and then stopped.Operate the mass spectrometer in APCI positive multiple reactionmonitoring mode. Use data from standard curve samples and generate alinear fit calibration curve for each analyte using the MultiQuan™software. Use the standard curve generated to calculate the analyteconcentrations for the unknowns.

Calculate compound IC₅₀ values using the mass spectrometric measurementof kynurenine from the media containing 500 μM of reference standardtreatment as one hundred percent inhibition, and no compound but DMSOtreatment as zero percent inhibition. Measurements ofN-formyl-kynurenine and tryptophan are used to assess the validity ofdata generated by showing direct relationship between kynurenine andN-formyl-kynurenine production with the concomitant reduction intryptophan levels. The results of this assay demonstrate that allexemplified compounds inhibit the production of kynurenine andN-formyl-kynurenine in IDO01 expressing cancer cells at IC₅₀ values forinhibiting both kynurenine and N-formyl-kynurenine of less than 0.9 μM.and of those tested (Examples 1-9) in cell viability, all of thecompounds did so without being overtly toxic to the cells up to at least1 μM. For example, the IC₅₀ for Example 1A for inhibiting kynurenine andN-formyl-kynurenine are 0.007 μM±0.002 (n=6) and 0.007 μM±0.002 (n=6)respectively. Furthermore, Example 1A does not inhibit cellproliferation up to 10 μM.

In Vivo Target Inhibition Assay

The purpose of this assay is to evaluate the inhibition of kynurenineproduction and tryptophan depletion in cancer cells in vivo. SKOV3X(Indiana University Research and Technology Center), an ovarian cancercell line, intrinsically expresses IDO1 and readily forms tumors in theperitoneal cavity of Athymic Nude-Foxn1^(nu) mice (Harlan). As aconsequence of IDO1 expression, SKOV3X tumors locally deplete tryptophanwith the concomitant production of high levels of kynurenine in thetumor microenvironment. The purpose of this assay is to measure theability of test compounds to inhibit IDO1 evidenced by the clearreduction in kynurenine levels in the tumor.

Live Phase

Culture SKOV3X in McCoys 5A media (Gibco, Cat #16600-082) supplementedwith non-essential amino acids (Gibco, Cat #11140-050), 1 mM sodiumpyruvate (Gibco, Cat #1360-070) and 10% FBS and incubate at 37° C. in 5%CO₂. Trypsinize and isolate cells from culture and resuspend cells inHank's balanced salt solution (HBSS). Implant 2×10⁶ SKOV3X cells intothe intraperitoneal cavity of each Athymic Nude-Foxn1^(nu) mouse(Harlan). Approximately three weeks post-implantation, palpate animalsto ensure tumor formation and randomize tumor-bearing mice into vehiclecontrol and compound treatment groups. Administer compound formulated invehicle containing 1% hydroxyethylcellulose (HEC) and 0.025% TWEEN® 80and 0.05% Antifoam or vehicle alone by oral gavage. Generate time-courseinhibition profile by dosing tumor-bearing animals with a single doseand collect plasma, liver, and tumor samples at 2, 4, 8, 12, and 24hours post dose. Collect blood into EDTA-containing blood collectiontubes (Greiner bio-one, Cat #450474) and centrifuge at 2365×g, isolateplasma, and freeze at −80° C. Isolate liver and tumor fragments, recordweights and flash freeze and store at −80° C.

Generation of Standard Curve, Tissue Processing and Target Inhibition

Prepare calibration standards for L-kynurenine and L-tryptophan by firstgenerating stripped matrices, which are plasma and tissue homogenatesdepleted of L-kynurenine and L-tryptophan by dialysis. Then, fortifystripped matrices with known amounts of L-kynurenine and L-tryptophan.Generate stripped mouse plasma by adding 10 mL of EDTA treated mouseplasma (BioreclamationIVT, Cat # MSEPLEDTA3) to a SPECTRA/POR®FLOAT-A-LYZER® G2 (Spectrum Labs, Cat # G235063) and placing thisdialysis device in 1000 mL of phosphate buffered saline and dialyzeovernight. Afterward, transfer this device to a fresh 1000 mL ofphosphate buffered saline and repeat the dialysis. Transfer the strippedmouse plasma to a clean container and store at −20° C. for future use.Prepare control liver homogenate by adding 3 mL of MeOH/water (1:1, v/v)for every gram of control mouse liver and homogenize with an ultrasonicprobe. Prepare control tumor homogenate in the same fashion except use atissue grinder to homogenize tumor tissue. Add 10 mL of the controltissue homogenates, liver and tumor, to separate SPECTRA/POR®FLOAT-A-LYZER® G2 devices and dialyze each overnight in 1000 mL ofMeOH/water (1:1, v/v), then transfer each to a fresh 1000 mL ofMeOH/water (1:1, v/v) and repeat the dialysis. Transfer the strippedtissue homogenates to separate containers and store at −20° C. forfuture use.

Prepare standard stock solutions of L-kynurenine-sulfate (Sigma Aldrich,Cat # K3750), dissolved in ACN/water (1:1, v/v) and L-tryptophan (SigmaAldrich), dissolved in N-methyl-2-pyrrolidone/water (4:1, v/v), to givefinal free base concentrations of 1 mg/mL Aliquot 50 μL of therespective stock solutions and dilute with MeOH/water (1:1, v/v) toyield a combined 50 μg/mL working solution. Prepare six additionalcalibration working solutions in MeOH/water (1:1, v/v) by serialdilution of the 50,000 ng/mL solution to obtain a 7-point calibrationcurve with final concentrations of 25 ng/mL to 50 μg/mL.

Mix liver samples acquired from test subjects with MeOH/water (1:1, v/v)in a proportion of 1 gram of tissue to 3 mL of solvent and homogenizedwith an ultrasonic probe. Homogenize tumor samples with the sameproportion of MeOH/water (1:1, v/v) using a tissue grinder. Thaw plasmasamples from test subjects and mix for homogeneity.

Perform extraction of calibration working solutions, the 7-pointdilution series of L-kynurenine and L-tryptophan, by transferring 25 μLof each sample to separate wells of a 96-well plate and add 25 μL of theappropriate stripped control matrix (plasma, liver or tumor homogenate)to these wells depending upon tissue of origin of test samples. Add 25μL of MeOH/water (1:1, v/v) to separate wells followed by 25 μL of therespective test samples. Next, add 180 μL of ACN/MeOH (1:1, v/v)containing 250 ng/mL of L-tryptophan-2′,4′,5′,6′,7′-d5 (Sigma Aldrich,Cat #615862) and L-kynurenine sulfate-ring-d4,3,3-d2 (Cambridge IsotopeLaboratories, Cat # DLM-7842-0.005) to all wells and mix to precipitateproteins in the samples. Centrifuge the 96-well plate to pellet theprecipitated protein material then dilute a portion of each supernatantat least 10-fold with water/TFA (100:2, v/v). Inject 10 μL of eachextracted sample and calibration standard onto an LC/MS-MS systemconsisting of a SHIMADZU® SCL-10A controller with SHIMADZU® LC-10ADvpHPLC pumps, a CTC-PAL autosampler and an AB SCIEX® 4000 triplequadrupole mass spectrometer. Separate the analytes on an Advantage™Echelon™ C18 column, 2.1×20 mm, 4 μm (Analytical Sales and Service, Cat# Sprite AE1822) maintained at ambient conditions with a mobile phaseflow rate of 1.5 mL/minute. Mobile phase A is water/TFA/1 M ammoniumbicarbonate, (1000:4:1, v/v/v) and mobile phase B is ACN/TFA/1 Mammonium bicarbonate 1000:4:1, v/v/v). The gradient profile is: 0minutes, 0.3% B; 0.03 to 0.2 minutes, 7% B; 0.3 to 0.4 minutes, 36% B;0.41 minutes, 98% B, then stopped at 0.7 minutes to return to theoriginal conditions. Operate the mass spectrometer in TURBOIONSPRAY®positive multiple reaction monitoring mode. Use data from calibrationstandards curve samples and generate a quadratic fit calibration curvefor each analyte using the Analyst™ software. Use the standard curvegenerated to calculate the analyte concentrations for the study samples.

Use the liver concentration of kynurenine from non-tumor-bearing animalstreated with vehicle as maximum inhibition or lowest level ofkynurenine. Use the SKOV3X tumor concentration of kynurenine fromvehicle-treated tumor-bearing mice as minimum inhibition or highestlevel of kynurenine. Calculate the percent inhibition for compoundtreated groups relative to the minimum IDO1 inhibition in thevehicle-treated tumor. The results of this assay demonstrate thatExample 1A inhibits the production of kynurenine and N-formyl-kynureninein IDO1 expressing cancer cells in vivo. Specifically, Example 1A dosedat 75 mg/kg, 25 mg/kg and 5 mg/kg resulted in 79%, 59% and 37%inhibition respectively 12 hours after dosing.

Anti-Tumor Effect of Example 1A in Mouse Syngeneic Colon26 Model forColon Cancer and in Combination with LY3300054 in Established L55Humanized Mouse Model

Mouse Syngeneic Colon 26 Model

Grow the mouse BALB/c-derived Colon26 colon cancer cell line in RPMI1640 medium supplemented with 10 mM HEPES, 1 nM sodium pyruvate, and 10%fetal bovine serum. Harvest sub-confluent cells with trypsin and rinsetwice with complete growth medium lacking serum. Initiate subcutaneoustumors by injecting 1×10⁶ cells resuspended in HBSS in the rear flank ofimmune-competent BALB/c mice (Envigo, Indianapolis, Ind.). Six daysafter tumor implantation, randomize animals based on body weight andplace into their respective treatment groups using the number of animalsper group as indicated.

L55 Humanized Tumor Model, hPBMC Challenge, and Treatment:

Grow the human NSCLC cell line, L55, in RPMI 1640 medium supplementedwith 10% fetal bovine serum. Harvest sub-confluent cells with trypsinand rinse twice with growth medium lacking serum. Initiate the growth ofsubcutaneous tumors by injecting 5×10⁶ in a 1:1 mixture of HBSS andMATRIGEL® (BD Biosciences, Franklin Lakes, N.J.) in the rear flank ofNOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ mice more commonly known as NODscid gamma chain knockout mice (NSG) mice (The Jackson Laboratory, BarHarbour, Me.), which lack T cells, B cells, NK cells, and are deficientin cytokine signaling. When the mean tumor volume reaches approximately200-300 mm³, randomize the animals by tumor size and body weight andplace into their respective treatment groups as indicated. Afterrandomization, challenge tumor-bearing mice with PBS alone (no PBMCs) orwith PBS containing 1×10⁷ human PBMCs into the tail vein of recipients.

Data Capture, Compound Formulation and Vehicle Controls (Both Models)

Capture tumor size and body weight using Study Director. Estimate tumorvolume (V) by using the formula: V=0.536×L×W² where L=larger measureddiameter and W=smaller of the perpendicular diameter. Transform thetumor volume data to a log scale to equalize variance across time andtreatment groups. Analyze the log volume data with a two-way repeatedmeasures analysis of variance by time and treatment using the MIXEDprocedures in SAS software (Version 9.2). The correlation model for therepeated measures is Spatial Power. Compare treated groups to thecontrol group at each time point. Use the MIXED procedure alsoseparately for each treatment group to calculate adjusted means andstandard errors at each time point. Both analyses account for theautocorrelation within each animal and for the loss of data that occurswhen animals with large tumors are removed from the study early.Calculate relative changes in tumor volume (% T/C) using the tumorvolume measurements taken nearest to the last day of dosing with Example1A, using the formula % T/C=100×ΔT/ΔC, where T=mean tumor volume of thecompound treated group, ΔT=mean tumor volume of the compound treatedgroup minus the mean tumor volume on the baseline day, C=mean tumorvolume of the control (vehicle) group, and ΔC=mean tumor volume of thecontrol group minus the mean tumor volume on the baseline day. If ΔT<0,then a tumor regression value is calculated instead of % T/C whereby %Regression=100×ΔT/T_(initial) such that T_(inital)=mean tumor volume onthe baseline day.

Assess antitumor efficacy of Example 1A and LY3300054 alone, or incombination by measuring tumor volume by three dimensional calipermeasurements twice a week during the course of the study. Measure bodyweight twice weekly during the course of the study, as a generalindicator of tolerability.

Formulations for Example 1A and LY3300054: Formulate Example 1A on aweekly basis in 1% HEC/0.25% Tween 80/0.05% Antifoam and store at 4° C.Solublize LY3300054 in phosphate buffered saline and store at 4° C.

Control group(s): For single agent efficacy studies, administer vehiclefor Example 1A alone. For combination studies, administer both vehiclesused for Example 1A and LY3300054 according to the same schedule foreach compound, respectively.

For monotherapy groups in combination efficacy studies, treat theanimals with the desired compound and the vehicle for the compound notbeing dosed following the schedule for the non-dosed compound.

Colon26 Syngeneic Model, Treatment and Results:

Monotherapy Example 1A

Treat female BALB/c mice (n=10) bearing Colon26 tumors with Example 1Atwice daily for 21 days by oral gavage at doses of 10, 50, and 100mg/kg. Start administration of Example 1A six days after tumorimplantation, and monitor tumor growth and body weight twice a week forthe duration of the treatment period.

Results:

Treatment with 10, 50, and 100 mg/kg of Example 1A resulted in adose-responsive effect on tumor growth with only 50 and 100 mg/kg dosesshowing statistically (p<0.001) relevant growth inhibition at day 20.The changes in tumor volume (% T/C) observed at day 20 were 17.5%,31.2%, and 62.6% for the 10, 50, and 100 mg/kg doses, respectively.There were no significant tolerability issues at any dose tested withExample 1A with respect to body weight changes over the course oftreatment compared to vehicle-treated mice. Body weight loss wasmeasured as the percent change from mean body weights recorded onbaseline 6 days after tumor implant for each group. At day 20, theaverage vehicle treated mice showed 5.5% reduction in body weightcompared to baseline with the 10, 50, and 100 mg/kg dosed groups showinga 2.5%, 8%, and 2.5% reduction, respectively. While there was adose-dependent trend in body weight loss with regard to dose, they werenot statistically different from vehicle-treated mice.

L55 Humanized Tumor Model, Treatment and Results:

hPBMC Effect on L55 Tumor Growth

The L55 NCLC human cancer cell line is intrinsically resistant to theallo-response associated with the injection of hPBMCs. The goal of thesestudies is to assess the ability of compounds to potentiate the alloresponse allowing human T cells to target and restrict the growth of ahuman L55 tumors in a mouse that lacks an adaptive immune system (NSGmice). To assess the contribution of hPBMCs on tumor growth inhibitionof the L55 tumors, mock inject NSG mice bearing established L55 tumors(n=10) that have reached approximately 250 mm³ with PBS lacking hPBMCs,or PBS containing 1×10⁷ hPBMCs. Measure tumor volume and body weighttwice a week for the duration of the treatment period.

Results:

There was no statistically significant inhibition of L55 tumor growthwhen compared to animals that did not receive hPBMCs over the course ofthe study. No significant tolerability issues were observed with theinjection of human PBMCs over the course of the study evidenced by thelack of significant weight loss when compared to baseline, which at day41 was 0.1% lower than at baseline.

Monotherapy Example 1A

To assess the ability of Example 1A to enhance L55 tumor growthinhibition mediated by hPBMCs, mock inject NSG mice bearing establishedL55 tumors (n=10) that have reached approximately 250 mm³ with PBSlacking hPBMCs, and another group (n=10) with PBS containing 1×10⁷hPBMCs. Treat both groups with 75 mg/kg Example 1A by oral gavage twicedaily for 21 days. Measure tumor volume and body weight twice a week forthe duration of the treatment period.

Results:

In the absence of hPBMCs, treatment of L55 tumors with Example 1A didnot result in significant tumor growth inhibition over the course of thetreatment when compared to vehicle alone without PBMCs. Treatment of L55tumor-bearing animals with Example 1A in the presence of hPBMCs resultedin tumor growth inhibition when compared to the vehicle control grouplacking hPBMCs. Statistically relevant suppression of tumor growth wasmost apparent at later time points with a % T/C of 47.6% at day 41(P<0.001). No significant tolerability issues were apparent over thecourse of the study with hPBMCs, Example 1A, or the combinationevidenced by the lack of statistically significant reductions in bodyweight loss when compared to baseline measurements.

Monotherapy LY3300054

To assess the ability of LY3300054 to enhance L55 tumor growthinhibition mediated by hPBMCs, inject two groups of NSG mice bearingestablished L55 tumors (n=10/group) that have reached approximately 250mm³ with PBS containing hPBMCs. Treat one group with 10 mg/kgIgG-effector null (IgG-EN) control antibody and the other with 10 mg/kgLY3300054 by intraperitoneal injection once a week for 4 weeks. Measuretumor volume and body weight twice a week for the duration of thetreatment period.

Results:

Treatment of L55 tumor-bearing mice that were injected with hPBMCs with10 mg/kg IgG-EN did not alter tumor growth or progression when comparedto vehicle alone with or without hPBMCs. Treatment of L55 tumor-bearinganimals that had been injected with hPBMCs with 10 mg/kg LY3300064resulted in statistically significant tumor growth inhibition whencompared to vehicle-treated controls that contained or lacked hPBMCs.The change in tumor volume (% T/C) observed at the end of the dosingperiod when compared to vehicle alone lacking PBMCs (day 37) was 75.7%.No significant tolerability issues were apparent over the course of thestudy with LY3300054 with or without hPBMCs evidenced by the lack ofstatistically significant reductions in body weight loss when comparedto baseline measurements.

Combination of Example 1A and LY3300054

Inject NSG mice (n=10) bearing L55 tumors that have reachedapproximately 250 mm³ with hPBMCs and treat with 75 mg/kg Example 1Atwice a day by oral gavage for 21 days and 10 mg/kg LY3300054 byintraperitoneal injection once a week for 4 weeks. Measure tumor volumeand body weight twice a week for the duration of the treatment period.

Results:

Combined treatment of 75 mg/kg Example 1A and 10 mg/kg LY3300054resulted in an improvement in the anti-tumor efficacy when compared toeither monotherapy group alone. Tumor volumes were significantly lowerthan the vehicle alone groups that either lacked PBMCs or were injectedwith hPBMCs (P<0.001 at all measurements). Tumor volumes on days 30, 34,37, and 41 were 9.6% T/C, 19.8% T/C, 13.3% T/C, and 27.3% T/C,respectively. The difference in the anti-tumor efficacy betweenmonotherapy groups compared to the combination group was statisticallysignificant (p<0.001). To assess whether or not the combination wasadditive or synergistic, the data is analyzed essentially as follows:

Statistical Analysis (Both Models):

The statistical analysis of the tumor volume data begins with a datatransformation to a log scale to equalize variance across time andtreatment groups. The log volume data are analyzed with a two-wayrepeated measures analysis of variance by time and treatment using theMIXED procedures in SAS software (Version 9.3). The correlation modelfor the repeated measures is Spatial Power. Treated groups are comparedto the control group at each time point. The MIXED procedure is alsoused separately for each treatment group to calculate adjusted means andstandard errors at each time point. Both analyses account for theautocorrelation within each animal and the loss of data that occurs whenanimals with large tumors are removed from the study early. The adjustedmeans and standard errors (s.e.) are plotted for each treatment groupversus time.

Combination Analysis Method (Bliss Independence for IVEF Studies):

With the results of the repeated measures analysis, contrast statementsare used to test for an interaction effect at each time point, comparingthe mean of the vehicle and combination groups to the mean of the twosingle agent groups. This is equivalent to the Bliss Independence methodfor testing additivity. The expected additive response (EAR) for thecombination is calculated on the tumor volume scale as: EARvolume=V1*V2/V0, where V0, V1, and V2 are the estimated mean tumorvolumes for the vehicle control, treatment 1 alone, and treatment 2alone, respectively. If the interaction test is significant, thecombination effect is declared statistically more than additive or lessthan additive depending on the observed combination mean volume beingless than or more than the EAR volume, respectively. Otherwise, thestatistical conclusion is additive.

Using this method of analysis, the tumor growth inhibition was notbetter than additive until days 34 and 37 where tumor growth inhibitionwas synergistic with P<0.008 and p<0.001, respectively. No significanttolerability issues were apparent over the course of the study with thecombination of Example 1A and LY3300054 evidenced by the lack ofstatistically significant reductions in body weight loss when comparedto baseline measurements.

The compounds of the present invention are preferably formulated aspharmaceutical compositions administered by a variety of routes. Mostpreferably, such compositions are for oral or intravenousadministration. Such pharmaceutical compositions and processes forpreparing same are well known in the art. See, e.g., REMINGTON: THESCIENCE AND PRACTICE OF PHARMACY (D. Troy, et al., eds., 21st ed.,Lippincott Williams & Wilkins, 2005).

As used herein, the term “effective amount” refers to the amount or doseof compound of the invention, or a pharmaceutically acceptable saltthereof which, upon single or multiple dose administration to thepatient, provides the desired effect in the patient under diagnosis ortreatment.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount for a patient, a number of factors areconsidered by the attending diagnostician, including, but not limitedto: the species of mammal; its size, age, and general health; thespecific disease or disorder involved; the degree of or involvement orthe severity of the disease or disorder; the response of the individualpatient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances.

The compounds of the present invention are generally effective over awide dosage range. For example, dosages per day normally fall within thedaily range of about 0.05-1000 mg. Preferably such doses fall within thedaily range of 0.1-500 mg. More preferably such doses fall within thedaily range of 1-200 mg. In some instances dosage levels below the lowerlimit of the aforesaid ranges may be more than adequate, while in othercases still larger doses may be employed without causing any harmfulside effect, and therefore the above dosage ranges are not intended tolimit the scope of the invention in any way. It will be understood thatthe amount of the compound actually administered will be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound or compounds administered, the age, weight, and response of theindividual patient, and the severity of the patient's symptoms.

SEQUENCE LISTING Amino Acid and Nucleotide SequencesSEQ ID NO: 1 (human PD-L1)MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEETSEQ ID NO: 2 (HCDR1 of LY3300054) KASGGTFSSYAISSEQ ID NO: 3 (HCDR2 of LY3300054) GIIPIFGTANYAQKFQGSEQ ID NO: 4 (HCDR3 of LY3300054) ARSPDYSPYYYYGMDVSEQ ID NO: 5 (LCDR1 of LY3300054) SGSSSNIGSNTVNSEQ ID NO: 6 (LCDR2 of LY3300054) YGNSNRPSSEQ ID NO: 7 (LCDR3 of LY3300054) QSYDSSLSGSVSEQ ID NO: 8 (HCVR of LY3300054)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSPDYSPYYYYGMDVWGQGTTVTVSS SEQ ID NO: 9 (LCVR of LY3300054)QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCQSYDSSLSGSV FGGGIKLTVLGSEQ ID NO: 10 (HC of LY3300054)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSPDYSPYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKSEQ ID NO: 11 (LC of LY3300054)QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCQSYDSSLSGSVFGGGIKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPAECSSEQ ID NO: 12 (DNA of HC of LY3300054)CAGGTCCAGCTGGTCCAGTCAGGGGCCGAGGTCAAAAAGCCAGGGTCATCTGTCAAAGTGTCTTGTAAGGCATCCGGGGGCACATTTTCCAGCTACGCTATCTCCTGGGTGAGACAGGCACCAGGGCAGGGTCTGGAGTGGATGGGCGGAATCATTCCCATCTTCGGGACCGCCAACTACGCTCAGAAGTTTCAGGGAAGGGTCACTATTACCGCCGACAAAAGCACATCTACTGCTTATATGGAGCTGTCTAGTCTGAGGTCTGAAGATACCGCAGTGTACTATTGCGCCCGGAGTCCCGACTATAGCCCTTACTATTACTATGGCATGGATGTCTGGGGCCAGGGAACCACAGTGACAGTCTCATCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAAGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCATCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATTCCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGCAAASEQ ID NO: 13 (DNA of LC of LY3300054)CAGTCCGTCCTGACACAGCCACCCTCAGCCTCTGGCACCCCTGGGCAGCGAGTGACAATCTCTTGTTCTGGGAGTTCCTCAAATATTGGTAGTAACACCGTGAATTGGTACCAGCAGCTGCCCGGCACAGCACCTAAGCTGCTGATCTATGGAAACTCAAATAGGCCATCCGGAGTCCCCGACCGGTTCTCTGGTAGTAAATCAGGCACTTCCGCCAGCCTGGCTATTAGCGGGCTGCAGTCTGAGGACGAAGCCGATTACTATTGCCAGTCTTACGATTCCAGCCTGTCTGGAAGTGTGTTTGGCGGAGGGATCAAGCTGACCGTCCTGGGCCAGCCTAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTCT

We claim:
 1. A method of treating a patient for cancer comprisingadministering a combination comprising4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamideand LY3300054 and wherein the cancer is non-small cell lung cancer orcolon cancer.
 2. The method of claim 1 wherein the cancer is non-smallcell lung cancer.
 3. The method of claim 1 wherein the cancer is coloncancer.
 4. The method of claim 1, wherein4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamideand LY3300054 are administered simultaneously, separately, orsequentially.
 5. The method of claim 1 wherein the4-fluoro-N-{(1R)-1-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)-2,3-dihydro-1H-indol-5-yl]ethyl}benzamideis in crystalline form characterized by an X-ray powder diffractionpattern (Cu radiation, λ-1.54060 Å) comprising at least one peak at17.38° in combination with one or more peaks selected from the groupconsisting of 12.51°, 15.65°, 16.37°, 17.56°, 21.48° and 25.23°(20±0.2°).